Compounds for modulating RNA binding proteins and uses therefor

ABSTRACT

The invention relates to compositions and methods for inhibiting RNA binding proteins (e.g., MEX-3, MEX-5 and POS-1), as well as methods for treating and preventing disorders associated with parasitic infections and inflammatory disorders.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/220,985, filed Jun. 26, 2009. The entire contents of theforegoing application are hereby incorporated in their entirety.

GOVERNMENT INTERESTS

This invention was made with government support under grant nos.1R21NS059380 and 3R21NS059380-01S1 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

SUBMISSION ON COMPACT DISC

This application incorporates by reference the ASCII text fileidentified by the name SequenceListing.txt, containing 1.49 KB of data,created on Nov. 12, 2010 and filed in computer-readable format (CRF).

BACKGROUND OF THE INVENTION

According to the World Health Organization (WHO), more than one-third ofthe world's population, approximately 2 billion people, is infested withhelminths. In 1999, the WHO estimated that schistosomiasis andsoil-based helminths represented more than 40% of the disease burden dueto all tropical disease, excluding malaria. While most parasiticinfestations are preventable and treatable, the effects of aninfestation can be chronic and long-term and may eventually cause death.For example, a person who has endured persistent and heavy infestationsis likely to suffer from anemia, malnutrition and chronic irreversiblediseases, such as liver fibrosis, cancer of the bladder and kidneyfailure. These parasites also affect livestock, which can facilitateinfestation in humans by causing contamination via soil or foodsupplies.

In addition to the health risk posed to humans by parasites, plants arehighly susceptible to parasitic infestations. Effects of nematode damageto plants include stunting, chlorosis, nutrient deficiencies, wilting,root abnormalities and reduced yield.

While there are a number of antihelminthic treatments currentlyavailable, some scientists are concerned that the parasites will developresistance to these treatments, especially in developing countries wherepeople are repeatedly infected with helminths and receive multiple dosesof antihelminthic drugs. In fact, resistance to antihelminthic drugs hasbeen observed in livestock due to frequent and repeated treatments.Moreover, rates of re-infection by helminths are very high due to thenumber and the durability of infective eggs on surfaces and in soil.While infection is active, some parasitic nematode species (e.g.,Ascaris lumbricoides) release an estimated 100,000 new embryos per adultfemale per day. Currently available anti-helminthics work by paralyzingthe infectious nematode through blocking ion channels and receptors anddo not inactivate the embryos that cause re-infection. Therefore, it isadvantageous to develop new therapies for the treatment and preventionof parasitic infestations and, in particular, therapies that limit thepossibility of parasitic re-infection, in both plants and animals,

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the development anduse of screening assays to identify compounds that inhibit theRNA-binding activity of MEX-3, MEX-5 and/or POS-1, RNA binding proteinswhich are required for early embryogenesis in parasitic worms. Theidentified compounds represent a novel class of anti-parasitic agentsthat specifically target parasitic worm embryos.

Accordingly, in one aspect, the invention pertains, at least in part, tomethod for treating or preventing a parasitic associated state in asubject comprising administering to the subject an effective amount ofan RNA binding modulatory compound (e.g., a compound of formula I, Ia,Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc,VIId, VIIe, VIIf or a compound of Table 1 or 2), such that the parasiticassociated state is treated or prevented. In one embodiment, theparasitic associated state is a parasitic infestation or re-infestation.In another embodiment, the parasitic associated state is a diseasecaused by a parasitic infestation.

In one embodiment, the invention pertains, at least in part, to a methodfor treating or preventing a parasitic infestation in a subject infestedwith or at risk for infestation with parasites by administering to thesubject a therapeutically or pesticidally effective amount of an RNAbinding modulatory compound, e.g., a compound of formula I, Ia, Ib, II,IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId,VIIe, VIIf or a compound of Table 1 or 2, such that the parasiticinfestation is treated or prevented.

In another embodiment, the invention pertains, at least in part, to amethod for protection of plants from a parasitic infestation byadministering to the plants a pesticidally effective amount of an RNAbinding modulatory compound, e.g., a compound of formula I, Ia, Ib, II,IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId,VIIe, VIIf or a compound of Table 1 or 2, such that the plants areprotected.

In yet another embodiment, the invention pertains, at least in part, toa method for inhibiting parasitic embryogenesis in a parasite or in asubject suffering from a parasitic infection by administering to theparasite or subject suffering from the parasitic infection atherapeutically or pesticidally effective amount of an RNA bindingmodulatory compound, e.g., a compound of formula I, Ia, Ib, II, IIa,IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe,VIIf or a compound of Table 1 or 2, such that embryogenesis isinhibited.

In a further embodiment, the invention pertains, at least in part, to amethod for reducing parasitic burden in soil, in plants or in an animalsuffering from a parasitic infection by administering to the soil,plants or animal a therapeutically or pesticidally effective amount ofan RNA binding modulatory compound, e.g., a compound of formula I, Ia,Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc,VIId, VIIe, VIIf or a compound of Table 1 or 2, such that the parasiticburden is reduced.

In another embodiment, the invention pertains, at least in part, to amethod for treating or preventing a disease caused by a parasiticinfestation in a subject by administering to the subject atherapeutically effective amount of an RNA binding modulatory compound,e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound ofTable 1 or 2, such that the disease is treated or prevented.

In one embodiment, the invention pertains, at least in part, to a methodfor treating or preventing an inflammatory disorder in a subject byadministering to the subject a therapeutically effective amount of anRNA binding modulatory compound, e.g., a compound of formula I, Ia, Ib,II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc,VIId, VIIe, VIIf or a compound of Table 1 or 2, such that theinflammatory disorder is treated.

In one embodiment, the subject is a plant. In one embodiment, thesubject is an animal. In one embodiment, the subject is a human.

In one embodiment, the parasite is present in a subject. In oneembodiment, the parasite is a helminth, e.g., a cestode, a trematode anda nematode. In one embodiment, the helminth is a nematode.

In one embodiment, the RNA binding modulatory protein modulates the RNAbinding activity of an RNA binding protein. In one embodiment, the RNAbinding protein is required for embryogenesis. In one embodiment, theRNA binding protein comprises a CCCH zinc finger motif. In oneembodiment, the RNA binding protein comprises a KH domain. In oneembodiment, the RNA binding protein is selected from the groupconsisting of MEX-5, POS-1 and MEX-3, or a homolog thereof. In oneembodiment, the RNA binding protein is MEX-5 or a homolog thereof. Inone embodiment, the RNA binding protein is MEX-3 or a homolog thereof.In one embodiment, the RNA binding protein is POS-1 or a homologthereof.

In another aspect, the invention pertains, at least in part, to apharmaceutical composition comprising a therapeutically effective amountof an RNA binding modulatory compound, e.g., a compound of formula I,Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb,VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, and apharmaceutically acceptable carrier.

In another aspect, the invention pertains, at least in a part, to acomposition comprising a pesticidally effective amount of an RNA bindingmodulatory compound, e.g., a compound of formula I, Ia, Ib, II, IIa,IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIe, VIId, VIIe,VIIf or a compound of Table 1 or 2, and an agronomically acceptablecarrier.

In yet another aspect, the present invention provides methods ofidentifying a compound useful in modulating a biological activity of anRNA-binding protein. The methods include providing an indicatorcomposition comprising an RNA-binding protein and an RNA moleculecomprising an RNA-binding protein recognition element; contacting theindicator composition with each member of a library of test compounds;determining the effect of the compound on a biological activity of theRNA-binding protein; and selecting a compound that modulates thebiological activity of the RNA-binding protein as compared to anappropriate control, thereby identifying a compound useful in modulatinga biological activity of an RNA-binding protein.

In another aspect, the invention provides methods of identifying acompound useful in modulating a biological activity of an RNA-bindingprotein. The methods include providing an indicator compositioncomprising an RNA-binding protein and an RNA molecule comprising anRNA-binding protein recognition element; contacting the indicatorcomposition with each member of a library of test compounds underconditions which allow binding of the RNA-binding protein to the RNAmolecule comprising an RNA-binding protein recognition element to form acomplex; and detecting the formation of a complex of the RNA-bindingprotein and the RNA molecule comprising an RNA-binding proteinrecognition element, wherein the ability of the compound to modulateinteraction of the RNA-binding protein and the RNA molecule comprisingan RNA-binding protein recognition element is indicated by a modulationof complex formation in the presence of the compound as compared to theformation of complex in the absence of the compound, thereby identifyinga compound useful in modulating a biological activity of an RNA-bindingprotein.

In one aspect, the present invention provides methods of identifying acompound useful in modulating embryogenesis. The methods includeproviding an indicator composition comprising an RNA-binding protein andan RNA molecule comprising an RNA-binding protein recognition element;contacting the indicator composition with each member of a library oftest compounds under conditions which allow binding of the RNA-bindingprotein to the RNA molecule comprising an RNA-binding proteinrecognition element to form a complex; and detecting the formation of acomplex of the RNA-binding protein and the RNA molecule comprising anRNA-binding protein recognition element, wherein the ability of thecompound to modulate interaction of the RNA-binding protein and the RNAmolecule comprising an RNA-binding protein recognition element isindicated by a modulation of complex formation in the presence of thecompound as compared to the amount of complex formed in the absence ofthe compound, thereby identifying a compound useful in modulatingembryogenesis.

In another aspect, the present invention provides methods of identifyinga compound useful for treating a subject with a parasitic-associatedstate. The methods include providing an indicator composition comprisingan RNA-binding protein and an RNA molecule comprising an RNA-bindingprotein recognition element; contacting the indicator composition witheach member of a library of test compounds under conditions which allowbinding of the RNA-binding protein to the RNA molecule comprising anRNA-binding protein recognition element to form a complex; and detectingthe formation of a complex of the RNA-binding protein and the RNAmolecule comprising an RNA-binding protein recognition element, whereinthe ability of the compound to modulate interaction of the RNA-bindingprotein and the RNA molecule comprising an RNA-binding proteinrecognition element is indicated by modulation of complex formation inthe presence of the compound as compared to the amount of complex formedin the absence of the compound, thereby identifying a compound usefulfor treating a subject with a parasitic-associated state.

The biological activity of the RNA-binding protein may be determined bymeasuring the interaction of the RNA-binding protein and an RNA-bindingprotein recognition element. Alternatively, the biological activity ofthe RNA-binding protein may be determined by determining the ability ofthe compound to modulate a biological activity selected from the groupconsisting of anterior patterning, germ cell totipotency, development ofthe intestine, development of germline blastomeres, development ofpharyngeal tissue, expression and/or activity of PAL-1, NOS-2, APX-1protein, and GLP-1.

The indicator composition may be a cell that expresses the RNA-bindingprotein or a cell-free composition.

In one embodiment, the RNA-binding protein comprises a CCCH-type tandemzinc finger. In one embodiment, the RNA-binding protein comprising theCCCH-type tandem zinc finger is POS-1.

In another embodiment, the RNA-binding protein comprises a KH domain. Inone embodiment, the RNA-binding protein comprising the KH domain isMEX-3.

In one embodiment, the RNA-binding protein recognition element comprisesthe consensus sequence UA(U₂₋₃)RD(N₁₋₃)G. In another embodiment, theRNA-binding protein recognition element comprises the consensus sequenceDKAG(N₀₋₃)UHUA.

Detecting the formation of a complex of the RNA-binding protein and theRNA molecule may be determined by a gel-shift assay or a fluorescencepolarization assay. In one embodiment, the RNA-binding proteinrecognition element is fluorescently labeled.

The methods of the invention may further comprise determining the effectof the test compound on a parasitic-associated state in a non-humananimal, comprising administering the test compound to the animal anddetermining the effect of test compound on the parasitic-associatedstate in the presence and absence of the test compound. Determining theeffect of the test compound on a parasitic-associated state may bedetermined by measuring an immune response in the non-human animal.

In one embodiment, the test compound increases the formation orstability of the complex. In another embodiment, the test compounddecreases the formation or stability of the complex.

In another aspect, the invention provides compounds identified accordingto the method of the invention which may be in the form of acomposition.

One aspect of the invention provides to methods of inhibitingembryogenesis in a parasite comprising contacting the parasite with acompound identified according to the methods of the invention.

In another aspect, the invention provides methods of treating aparasite-associated state in a subject, comprising administering acomposition comprising a compound identified in the methods of theinvention to the subject, thereby treating a parasite-associated statein the subject.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains generally to a method for treating or preventinga parasitic associated state in a subject comprising administering tothe subject an effective amount of an RNA binding modulatory compound(e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound ofTable 1 or 2), such that the parasitic associated state is treated orprevented.

The term “parasitic associated state” includes those diseases anddisorders associated with parasites. In one embodiment, the parasiticassociated state is a parasitic infestation. In one embodiment, theparasitic associated state is a disease caused by a parasiticinfestation. In one embodiment, the parasitic associated state is aparasitic re-infestation.

As used herein, the term “RNA binding modulatory compound” includesthose compounds that are capable of modulating the activity of anRNA-binding protein (e.g., an RNA-binding protein required forembryogenesis in a parasitic worm), e.g., the binding of an RNA-bindingprotein to a target RNA (e.g., an RNA molecule comprising an RNA-bindingrecognition element of the RNA-binding protein). In one embodiment, theRNA binding modulatory compound is a compound of formula I, Ia, Ib, II,IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId,VIIe, VIIf or a compound of Table 1 or 2. In one embodiment, the RNAbinding modulatory compound includes compounds that are capable ofdecreasing, inhibiting or preventing the activity of an RNA-bindingprotein, e.g., the binding of an RNA-binding protein to a target RNA(e.g., an RNA molecule comprising an RNA-binding recognition element ofthe RNA-binding protein). In another embodiment, the RNA bindingmodulatory compound includes compounds that are capable of increasing,augmenting or enhancing the activity of an RNA-binding protein, e.g.,the binding of an RNA-binding protein to a target RNA (e.g., an RNAmolecule comprising an RNA-binding recognition element of theRNA-binding protein).

In various embodiments, the method includes treating a parasiticinfestation in a subject infested with parasites, protecting plants froma parasitic infestation, inhibiting embryogenesis in a parasite or in asubject suffering from a parasitic infestation, reducing parasiticburden in soil, in plants or in a mammal suffering from a parasiticinfection, treating or preventing an inflammatory disorder and treatinga disease in a mammal caused by the growth and replication of aparasite.

In one embodiment, the invention pertains to a method of treating aparasitic infestation in a subject infested with parasites byadministering to the subject a therapeutically or pesticidally effectiveamount of an RNA binding modulatory compound, e.g., a compound offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, suchthat the subject is treated.

The term “subject” includes animals (e.g., vertebrates, amphibians,fish, mammals, non-human animals), including humans, that are capable ofsuffering from a parasitic infestation, an inflammatory disorder (e.g.,rheumatoid arthritis, psoriasis and multiple sclerosis) or a disease ina mammal caused by a parasitic infection (e.g., helminthiasis). Subjectsalso include primates, such as chimpanzees, monkeys and the like. In oneembodiment of the invention, the subject is suffering from a parasiticinfestation or infection, e.g., a helminth infestation. In oneembodiment, the subject is at risk for a parasitic infection, e.g., hasbeen exposed to a parasite e.g., a helminth.

The term “subject” also includes agriculturally productive livestock,for example, cattle, sheep, goats, horses, pigs, donkeys, camels,buffalo, rabbits, chickens, turkeys, ducks, geese and bees; and domesticpets, for example, dogs, cats, caged birds and aquarium fish, and alsoso-called test animals, for example, hamsters, guinea pigs, rats andmice.

The term “subject” also includes plants. The term “plant” includes allplants and plant parts, for example, all plants and plant populations,including wild plants or crop plants (including naturally occurring cropplants). Crop plants may be plants that can be obtained by conventionalplant breeding and optimization methods or by biotechnological andrecombinant methods or by combinations of these methods, includingtransgenic plants. Examples of plants include, but are not limited tothe following plant cultivars: cereals (wheat, barley, rye, oats, rice,maize, sorghum and related species); beet (sugar beet and fodder beet);pome, stone and berry fruit (apples, pears, plums, peaches, almonds,cherries, strawberries, raspberries and blackberries); legumes (beans,lentils, peas, soya); oil crops (rape, mustard, poppy, olives,sunflowers, coconut, castor oil, cocoa, peanut); cucumber plants(squashes, cucumber, melons); citrus fruits (oranges, lemons,grapefruits, mandarines); vegetables (spinach, lettuce, asparagus,cabbage varieties, carrots, onions, tomatoes, potatoes, paprika);laurels (avocado, cinnamonium, camphor) and plants such as tobacco,cotton, nuts, corn, coffee, aubergines, sugar cane, tea, pepper, vines,hops, grapes, bananas and natural rubber plants, as well as ornamentalplants. In one embodiment, the plant is suffering from a parasiticinfestation. In another embodiment, the plant is at risk of sufferingfrom a parasitic infestation.

Plant parts also include all parts and organs of plants above and belowthe ground, such as shoot, leaf, flower and root, examples of whichinclude, for example, leaves, needles, stalks, stems, flowers, fruitbodies, fruits, seeds, roots, tubers and rhizomes. The plant parts alsoinclude harvested material, and vegetative and generative propagationmaterial, for example cuttings, tubers, rhizomes, offsets and seeds.

The language “subject” also includes soil. The term “soil” includes thesoil used in planting any plants or plant parts as described above. Theterm “soil” also includes that soil that has not yet been planted withany plants or plant parts. In one embodiment, the soil suffers from aparasitic infestation.

As used herein, the term “treating” may result in prevention of thedisease or condition, cure of the disease or condition, a decrease inthe type or number of symptoms associated with the condition, either inthe long term or short term (i.e., amelioration of the condition) orsimply a transient beneficial effect to the subject.

The terms “treating” and “treatment” used in the context of an animalinclude the administration of a therapeutically effective amount of aRNA binding modulatory compound, e.g., compound of formula I, Ia, Ib,II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc,VIId, VIIe, VIIf or a compound of Table 1 or 2, to treat the animal fora parasitic associated state, e.g., parasitic infestation, parasiticembryogenesis, an inflammatory disease (e.g., rheumatoid arthritis,psoriasis and multiple sclerosis) or a disease in an animal caused by aparasitic infection (e.g., helminthiasis).

The terms “preventing” and “prevention” used in the context of an animalinclude the administration of a therapeutically or prophylacticallyeffective amount of a RNA binding modulatory compound, e.g., compound offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, toprevent a parasitic associated state, e.g., parasitic infestation,parasitic embryogenesis, an inflammatory disease (e.g., rheumatoidarthritis, psoriasis and multiple sclerosis) or a disease in an animalcaused by a parasitic infection (e.g., helminthiasis), from occurring.

The terms “treating” and “treatment” used in the context of plantsinclude the administration to a plant or to soil a pesticidallyeffective amount of a RNA binding modulatory compound, e.g., compound offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, totreat the plant or soil for a parasitic associated state, e.g.,parasitic infestation, parasitic embryogenesis or reduction of theparasitic burden of the plant or soil.

The terms “preventing” and “prevention” used in the context of plantsinclude the administration of a pesticidally effective amount of a RNAbinding modulatory compound, e.g., compound of formula I, Ia, Ib, II,IIa, IIc, IIb, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId,VIIe, VIIf or a compound of Table 1 or 2, to a plant or to soil toprevent a parasitic associated state, e.g., parasitic infestation orparasitic embryogenesis.

The phrases “parasitic infestation” and “parasitic infection”, usedinterchangeable herein, are intended to include the presence ofparasitic organisms or embryos within the subject (e.g., mammal, plantor soil). The term “parasite” includes an animal or plant that lives inor on a host subject (e.g., a mammal, plant or soil). In one embodiment,the parasite is a helminth. The term “helminth” includes eukaryotic,worm-like parasites that live inside their hosts. Examples of helminthsinclude, but are not limited to, flatworms (e.g., plathyhelminths) forexample, trematodes (e.g., flukes) and cestodes (e.g., tapeworms);thorny-headed worms (e.g., acanthocephalans); and roundworms (e.g.,nematodes). In one embodiment, the parasitic infestation is a helminthinfestation. In one embodiment, the parasitic infestation is a nematodeinfestation. In one embodiment, the helminth or parasitic worm is anintestinal parasite, e.g., a parasitic worm that lives inside thedigestive tract. As used herein, the phrase “parasitic re-infestation”or “parasitic re-infection” is intended to include the re-occurrence ofa parasitic infestation in a subject.

Examples of trematodes include, for example, Schistosoma spp. (e.g.,Schistosoma bovis, Schistosoma curassoni, Schistosoma edwardiense,Schistosoma guineensis, Schistosoma haematobium, Schistosoma hippotami,Schistosoma incognitum, Schistosoma indicum, Schistosoma intercalatum,Schistosoma japonicum, Schistosoma lieperi, Schistosoma malayenesis,Schistosoma mansoni, Schistosoma margrebowiei, Schistosoma mattheei,Schistosoma mekongi, Schistosoma ovuncatum, Schistosoma nasale,Schistosoma rodhaini, Schistosoma sinesium, Schistosoma spindale,Schistosoma sinensium), Trichobilharzia regenti, Clonorchis sinensis,Dicrocoelium dendriticum, Dicrocoelium hospes, Fasciola hepatica,Fascioloides magna, Fasciola giganta, Fasciola jacksoni, Metorchisconjunctus, Metorchis albidus, Protofasciola robusta, Parafasciolopsisfasciomorphae, Opisthorchis viverrini, Opisthorchis felineus,Opisthorchis guayaquilensis, Paragonimus westermani, and Fasciolopsisbuski).

Examples of cestodes include, for example, cyclophillidea (e.g.,Dipylidium caninum, Taenia crassiceps, Taenia hydatigena, Taeniamulticeps, Taenia pisiformis, Taenia serialis, Taenia taeniaeformis,Echinococcus granulosus, Echinococcus multilocularis, Echinococcusshiquicus, Echinococcus oligarthrus, Echinococcus vogeli, Echinococcusortleppi, Echinococcus equinus, Taenia saginata, Taenia solium,Hymenolepis nana, Hymenolepis diminuta) and pseudophyllidea (e.g.,Diphyllobothrium latum, Diphyllobothrium pacificum, Diphyllobothriumcordatum, Diphyllobothrium ursi, Diphyllobothrium dendriticum,Diphyllobothrium lanceolatum, Diphyllobothrium dalliae, Diphyllobothriumyonagoensis, Diphyllobothrium mansonoides, Spirometra erinaceieuropaei,Spirometra mansonoides).

Examples of nematodes include, for example, Dracunculus medinensis,Onchocerca volvulus, Loa loa, Mansonella perstans, Mansonella ozzardi,Mansonella streptocera, Dirofilaria immitis, Dirofilaria repens,Acanthocheilonema viteae, Brugia malayi, Brugia pahangi, Brugia timori,Cercopithifilaria johnstoni, Dipetalonema reconditum, Dipetalonemarepens, Dirofilaria immitis, Dirofilaria repens, Dirofilaria tenuis,Dirofilaria ursi, Elaeophora abramovi, Elaeophora bohmi, Elaeophoraelaphi, Elaeophora poeli, Elaeophora sagitta, Elaeophora schneideri,Foleyella furcata, Litomosa westi, Litomosoides brasiliensis,Litomosoides sigmodontis, Litomosoides wilsoni, Ochoterenelladigiticauda, Onchocerca gibsoni, Onchocerca gutturosa, Onchocercavolvulus, Piratuba digiticauda, Sarconema eurycerca, Waltonellaflexicauda, Wuchereria bancrofti, Wuchereria kalimantani, Gnathostomabinucleatum, Gnathostoma doloresi, Gnathostoma hispidum, Gnathostomalamothei, Gnathostoma malaysiae, Gnathostoma nipponicum, Gnathostomaspinigerum, Gnathostoma turgidum, Ancylostoma brazilienese, Ancylostomacaninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostomapluridenatum, Ancylostoma tubaeforme, Necator americanus,Angiostrongylus cantonensis, Mermis nigrescens, Trichuris trichiura,Trichinella spiralis, Caenorhabditis elegans, Strongyloides stercoralis,Micronema (Halicephalobus) delatrix, Haemonchus contortus, Ostertagiasp., Nematodirus sp., Nippostrongylus brasiliensis, Heligmosomoidespolygyrus, Dictyocaulus viviparous, Toxocara canis, Anisakis sp.,Enterobius sp., Thelazia sp., Ascaris lumbricoides, Ascaris suum,Anisakis pegreffi, Anisakis physeteris, Anisakis schupakovi, Anisakissimplex, Anisakis typical, Anisakis ziphidarum, Toxocara cati,Baylisacaris procyonis, Baylisacaris melis, Baylisacaris transfuga,Baylisacaris columnaris, Baylisacaris devosi, Baylisacaris laevis,Strongyloides stercoralis, Enterobius vermicularis, Enterobiusanthropopitheci, Enterobius gregorii, Trichinella spiralis, Trichuristrichiura, Trichocephalus trichiuris, Capillaria philippinensis,Belonolaimus anama, Belonolaimus euthychilus, Belonolaimus gracilis,Belonolaimus jara, Belonolaimus lineatus, Belonolaimus lolii,Belonolaimus longicaudatus, Belonolaimus maritimus, Belonolaimusnortoni, Criconemoides sp., Helicotylenchus digonicus, Helicotylenchuslabiodiscinus, Helicotylenchus leiocephalus, Helicotylenchus platyurus,Helicotylenchus pseudorobustus, Heterodera zeae, Hoplolaimustylenchiformis, Hoplolaimus galeatus, Hoplolaimus columbus, Xiphinemaamericanumv, Longidorus elongatus, Longidorus breviannulatus,Meloidogyne chitwoodi, Meloidogyne graminis, Meloidogyne hapla,Meloidogyne mayaguenesis, Meloidogyne partityla, Pratylenchus agilis,Pratylenchus alleni, Pratylenchus coffeae, Pratylenchus convallariae,Pratylenchus crenatus, Pratylenchus flakkensis, Pratylenchus hexincisus,Pratylenchus loosi, Pratylenchus penetrans, Pratylenchuspseudopratensis, Pratylenchus scribneri, Pratylenchus thornei,Trichodorus obtusus, Trichodorus proximus, Tylenchorhynchus cylindricus,Tylenchorhynchus hordei, Tylenchorhynchus nudus, Tylenchorhynchusrobustus, Globodera pallida, Globodera rostochiensis, Globoderaachilleae, Globodera artemisiae, Globodera chaubattia, Globoderahypolysi, Globodera leptonepia, Globodera millefolii, Globoderamirabilis, Globodera pseudorostochiensis, Globodera tabacumsolanacearum, Globodera tabacum tabacum, Globodera tabacum virginae,Globodera zelandica, Ditylenchus destructor, Heterodera glycines,Heterodera schachtii, Nacobbus aberrrans, Criconemella inusitatus,Bursaphelenchus xylophilis, Radopholus simila, Rotylenchulus reniformis,Tylenchulus semipenetrans, Belonolaimus longicaudatus, Macroposthoniacurvata, Macroposthonia discus, Macroposthonia annulata, Macroposthoniarustica, Macroposthonia sphaerocephalus, Macroposthonia xenoplax,Aphelenchoides besseyi, Aphelenchoides bicaudatus, Aphelenchoidescentralis, Aphelenchoides clarus, Aphelenchoides confusus,Aphelenchoides dactylocercus, Aphelenchoides obtusus, Aphelenchoidesparietinus, Aphelenchoides pusillus, Aphelenchoides sacchari,Aphelenchoides vigor and Ditylenchus dipsaci. In one embodiment, thenematode is C. elegans.

The language “therapeutically effective amount” of the compound includesthat amount necessary or sufficient to treat, prevent or ameliorate adisease or disorder (e.g., a parasitic associated state, e.g., parasiticinfestation) in a subject (e.g., a subject suffering from or at risk fora parasitic infestation, e.g., infestation by a helminth). Atherapeutically effective amount of the compound includes that amountnecessary or sufficient to treat, prevent or ameliorate in a subject aparasitic associated state, an inflammatory disorder (e.g., rheumatoidarthritis, psoriasis or multiple sclerosis) or a disease in a subjectcaused by a parasitic infestation (e.g., helminthiasis). The language“therapeutically effective amount” of the compound also includes thatamount necessary to reduce the parasitic burden in a subject and theamount necessary to inhibit parasitic embryogenesis in a parasite or ina subject suffering from a parasitic infestation. The therapeuticallyeffective amount can vary depending on such factors as the size andweight of the subject, the type of illness, etc. One of ordinary skillin the art would be able to study the aforementioned factors and makethe determination regarding the effective amount of the compoundswithout undue experimentation.

The language “pesticidally effective amount” of the compound includesthat amount necessary or sufficient to treat, prevent or ameliorate adisease or disorder (e.g., parasitic associated state, e.g., parasiticinfestation) in a plant or soil (e.g., plant or soil suffering from orat risk for a parasitic associated state, e.g., parasitic infestation).The language “pesticially effective amount” of the compound alsoincludes that amount necessary to reduce the parasitic burden in thesoil or plant and the amount necessary to inhibit parasiticembryogenesis in a parasite or in a plant or in the soil comprising aparasitic infestation. The pesticidally effective amount can varydepending on such factors as the type of plant, the type of parasiticinfestation, the extent of the parasitic infestation, etc. One ofordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the pesticidally effectiveamount of the compounds without undue experimentation.

In another embodiment, the invention pertains, at least in part, to amethod for protection of plants from a parasitic infestation byadministering to the plants a pesticidally effective amount of a an RNAbinding modulatory compound, e.g., compound of formula I, Ia, Ib, II,IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId,VIIe, VIIf, or a compound of Table 1 or 2, such that the plants areprotected.

The language “protect” and “protection” includes shielding, guarding orpreventing plants from damage by a parasitic infestation in or on theplant or in the soil surrounding the plant. The protection can occur byapplication of a an RNA binding modulatory compound, e.g., compound offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, to theplant itself or to the soil surrounding the plant.

In yet another embodiment, the invention pertains, at least in part, toa method for modulating, e.g., inhibiting, embryogenesis, e.g., in aparasite or in a subject suffering from a parasitic infection, bycontacting the parasite or administering to the subject suffering from aparasitic infection a therapeutically effective amount of an RNA bindingmodulatory compound, e.g., a compound of formula I, Ia, Ib, II, IIa,IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe,VIIf or a compound of Table 1 or 2, such that embryogenesis isinhibited. The parasite may be located externally from a plant (e.g., ona plant or in the soil) or may be located within the plant or mammalitself.

As used herein, the various forms of the term “modulate” includestimulation (e.g., increasing or upregulating a particular response oractivity) and inhibition (e.g., decreasing or downregulating aparticular response or activity).

The terms “inhibit” and “inhibiting” refer to decreasing ordownregulating a particular response or activity. The terms “inhibit”and “inhibiting” include, for example, the suppression or ameliorationof embryogenesis, e.g., parasitic embryogenesis. The terms “inhibit” and“inhibiting” also include, for example, the downmodulating or blockingof the interaction between an RNA binding protein and a target RNA.

The language “embryogenesis” includes the process by which an embryo,e.g., a parasitic embryo, is formed and develops, e.g., in a parasite.Without being bound by theory, in one embodiment, the compounds offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2modulate, e.g., inhibit, the binding of RNA binding proteins required inthe early stages of embryogenesis to a target RNA of the RNA bindingprotein. In one embodiment, the RNA binding protein is MEX-5, POS-1 orMEX-3.

The term “RNA binding protein,” as used herein, refers to a protein thatselectively or specifically binds to RNA. In one embodiment, the RNAbinding protein binds selectively to single-stranded RNA. In oneembodiment, the RNA binding protein binds selectively to double-strandedRNA. An RNA binding protein of the present invention selectively orspecifically binds to a target RNA, e.g., RNA that comprises an RNAbinding recognition element for the RNA binding protein.

In one embodiment, the RNA binding protein is required forembryogenesis, e.g., parasitic embryogenesis (e.g., helminthembryogenesis). In one embodiment, the RNA binding protein is a helminthRNA binding protein, e.g., a nematode RNA binding protein, a trematodeRNA binding protein or a cestode RNA binding protein. In one embodiment,the RNA binding protein is a mammalian RNA binding protein. In oneembodiment, the RNA binding protein is a mammalian RNA binding proteinand is not required for embryogenesis. In one embodiment of theinvention, the RNA binding protein comprises a CCCH Zinc finger motif.Examples of RNA binding proteins that comprise a CCCH Zinc finger motifinclude, but are not limited to, MEX-5, POS-1 and MEX-6 or a homologthereof. In another embodiment, the RNA binding protein comprises a KHdomain. Examples of RNA binding proteins that comprise a KH domaininclude, but are not limited to, MEX-3 and GLD-1, or a homolog thereof.

The term “MEX-5” includes a cytoplasmic RNA binding protein thatcontains two CCCH zinc finger motifs that functions along with thesimilar CCCH zinc finger protein MEX-6, and is necessary for transducingpolarity cues and establishing soma/germline asymmetry in the earlyembryo. In regulating the soma/germline asymmetry, MEX-5 activates theSOCS-box protein ZIF-1, which functions as part of an E3 ubiquitinligase complex that degrades germ plasm proteins in somatic blastomers,resulting in reduced expression of germline proteins in germlineblastomers. MEX-5 is expressed at uniform levels in both oocytes andnewly fertilized eggs (see Schubert C. M. et al., Molecular Cell (2000)5:671-682; Hunter, C. et al. Development (2002) 129:747-759; Cuenca, A.A. et al. (2003) Development 130:1255-1265; Lin Developmental Biology(2003) 258:226-239; DeRenzo C. et al., (2003) Nature 424:685-689; Nishiet al. (2008) Development 135:687-697).

The term “POS-1” includes a CCCH-type zinc finger protein that isnecessary for the proper fate specification of germ cells, intestine,pharynx and hypodermis. POS-1 is also required in posterior blastomersfor positive regulation of apx-1 mRNA translation and negativeregulation of glp-1 mRNA translation by direct binding to the spatialcontrol region in the glp-1 mRNA 3′ UTR. POS-1 is first found in lowlevels in one-cell embryos and in high levels in germline blastomeres,where it disappears after the P4 division. POS-1 colocalizes withcytoplasmic and perinuclear P granules in the germline blastomeres P1,P2, P3 and P4 (see Kohara, Y. et al. (1999) Development 126:1-11; Oguro,K. et al. (2003) Development 130:2495-2503).

The term “MEX-3” includes a KH domain-containing RNA binding proteinthat is necessary for specifying the identities of the anterior ABblastomer and its descendent, as well as for the identity of the P3blastomer and proper segregation of the germline P granules. MEX-3 isfound uniformly in the cytoplasm of oocytes and one-cell stage embryosand becomes more abundant in AB and its daughters at the two and fourcell stages (see Bowerman et al. (1996) Cell 87:205-216).

In one embodiment, the RNA binding modulatory compounds of theinvention, e.g., the compounds of formula I, Ia, Ib, II, IIa, IIb, IIc,III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or acompound of Table 1 or 2, modulate (e.g., inhibit) the RNA bindingactivity of MEX-5, but do not substantially inhibit the RNA bindingactivity of MEX-3 or POS-1. In another embodiment, the RNA bindingmodulatory compounds of the invention, e.g., the compounds of formula I,Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb,VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, modulate (e.g.,inhibit) the RNA binding activity of MEX-3, but do not substantiallyinhibit the RNA binding activity of MEX-5 or POS-1. In yet anotherembodiment, the RNA binding modulatory compounds of the invention, e.g.,the compounds of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI,VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table1 or 2, modulate (e.g., inhibit) the RNA binding activity of POS-1, butdo not substantially inhibit the RNA binding activity of MEX-3 or MEX-5.In a further embodiment, the RNA binding modulatory compounds of theinvention, e.g., the compounds of formula I, Ia, Ib, II, IIa, IIb, IIc,III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or acompound of Table 1 or 2 modulate (e.g., inhibit) the RNA bindingactivity of two of MEX-3, MEX-5 and POS-1, without substantiallymodulating (e.g., inhibiting) the RNA binding activity of the thirdprotein. In one embodiment, the RNA binding modulatory compounds of theinvention, e.g., the compounds of formula I, Ia, Ib, II, IIa, IIb, IIc,III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or acompound of Table 1 or 2, modulate (e.g., inhibit) the RNA bindingactivity of MEX-3, MEX-5 and POS-1. In one embodiment, the RNA bindingmodulatory compounds of the invention, e.g., the compounds of formula I,Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb,VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, modulate (e.g.,inhibit) the RNA binding activity of parasitic MEX-3, MEX-5 and/or POS-1without modulating (e.g., inhibiting) the RNA binding of the mammalian(e.g., human) homologs of MEX-3, MEX-5 and/or POS-1.

In one embodiment, an RNA binding modulatory compound of the invention,e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound ofTable 1 or 2, modulates, e.g., inhibits, the RNA binding activity of anRNA binding protein (e.g., MEX-3, MEX-5 and/or POS-1) by at least about75%, by about 76%, by about 77%, by about 78%, by about 79%, by about80%, by about 81%, by about 82%, by about 83%, by about 84%, by about85%, by about 86%, by about 87%, by about 88%, by about 89%, by about90%, by about 91%, by about 92%, by about 93%, by about 94%, by about95%, by about 96%, by about 97%, by about 98%, by about 99% or by about100%. In one embodiment, an RNA binding modulatory compound of theinvention, e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc,III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or acompound of Table 1 or 2, modulates, e.g., inhibits, the RNA bindingactivity of an RNA binding protein (e.g., MEX-3, MEX-5 and/or POS-1) byabout 25%, by about 30%, by about 35%, by about 45%, by about 50%, byabout 55%, by about 60%, by about 65%, by about 70%, by about 71%, byabout 72%, by about 73%, or by about 74%. In a preferred embodiment, anRNA binding modulatory compound of the invention, e.g., a compound offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2,modulates, e.g., inhibits, the RNA binding activity of an RNA bindingprotein (e.g., MEX-3, MEX-5 and/or POS-1) by at least about 75%.

In a further embodiment, the invention pertains, at least in part to amethod for reducing parasitic burden in soil, in plants or in a subjectsuffering from a parasitic infection by administering a therapeuticallyor pesticidally effective amount of an RNA binding modulatory compound,e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound ofTable 1 or 2, such that the parasitic burden is reduced.

The term “parasitic burden” includes the amount (number) of parasitesand/or parasite eggs present in a given sample. Appropriate samplesinclude, but are not limited to, soil samples, plant samples (e.g., fromroots, leaves, stems and the like) and biological samples (e.g., urine,blood, tissue, fecal matter and the like). In one embodiment, uponadministration of an RNA binding modulatory compound, e.g., a compoundof formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, to thesubject, the parasitic burden in that subject is reduced by about 10%,by about 11%, by about 12%, by about 13%, by about 14%, by about 15%, byabout 16%, by about 17%, by about 18%, by about 19%, by about 20%, byabout 21%, by about 22%, by about 23%, by about 24%, by about 25%, byabout 26%, by about 27%, by about 28%, by about 29%, by about 30%, byabout 31%, by about 32%, by about 33%, by about 34%, by about 35%, byabout 36%, by about 37%, by about 38%, by about 39%, by about 40%, byabout 41%, by about 32%, by about 43%, by about 44%, by about 45%, byabout 46%, by about 47%, by about 48%, by about 49%, 50%, by about 51%,by about 52%, by about 53%, by about 54%, by about 55%, by about 56%, byabout 57%, by about 58%, by about 59%, by about 60%, by about 61%, byabout 62%, by about 63%, by about 64%, by about 65%, by about 66%, byabout 67%, by about 68%, by about 69%, by about 70%, by about 71%, byabout 72%, by about 73%, by about 74%, by about 75%, by about 76%, byabout 77%, by about 78%, by about 79%, by about 80%, by about 81%, byabout 82%, by about 83%, by about 84%, by about 85%, by about 86%, byabout 87%, by about 88%, by about 89%, by about 90%, by about 91%, byabout 92%, by about 93%, by about 94%, by about 95%, by about 96%, byabout 97%, by about 98%, by about 99% or by about 100%.

In one embodiment, the invention pertains, at least in part, to a methodfor treating or preventing an autoimmune disorder or inflammatorydisorder in a subject by administering to the subject a therapeuticallyeffective amount of an RNA binding modulatory compound, e.g., a compoundof formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, suchthat the inflammatory disorder is treated.

The term “inflammatory disorder” includes those disorders that areassociated with acute or chronic inflammation. Examples of inflammatorydisorders include, but are not limited to, allergic reactions,autoimmune disorders (e.g., multiple sclerosis, diabetes, e.g., insulindependent diabetes mellitus, chronic obstructive pulmonary disease,lupus, endometriosis, myasthenia gravis, psoriasis, psoriaticarthritis), cancer, atherosclerosis, ischemic heart disease, asthma,autoimmune diseases, chronic inflammation, chronic prostatitis,glomerulonephritis, inflammatory bowel disease, pelvic inflammatorydisease, e.g., Crohn's disease, reperfusion injury, rheumatoidarthritis, ankylosing spondylitis, transplant rejection and vasculitis.

In another embodiment, the invention pertains, at least in part, to amethod for treating a disease caused by a parasitic infestation in amammal by administering to the mammal a therapeutically effective amountof an RNA binding modulatory compound, e.g., a compound of formula I,Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb,VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, such that thedisease is treated.

In one embodiment, the disease caused by a parasitic infestation ishelminthiasis. The term “helminthiasis” includes diseases caused by theinfestation of a mammal by the a helminth (e.g., a plathyhelminth, forexample, trematodes and cestodes; thorny-headed worms, for example,acanthocephalans; and roundworms, for example, nematodes). Examples ofheminthiasis include, but are not limited to, schistosomiasis, swimmer'sitch, clonorchiasis, fasciolosis, paragonimiasis, fasciolopsiasis,echinococcosis, taeniasis, cysticercosis, hymenolepiasis,diphyllobothriasis, sparganosis, dracunculiasis, onchocerciasis, loa loafilariasis, mansonelliasis, dirofilariasis, gnathostomiasis,ancylostomiasis, cutaneous larva migrans, necatoriasis,angiostrongyliasis, ascariasis, anisakiasis, viceral larva migrans,toxocariasis, strongyloidiasis, enterobiasis, pinworm, trichinosis,trichuriasis, whipworm and capillariasis.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “compound” or “test compound” includes anyagent, e.g., nucleic acid molecules, antisense nucleic acid molecule,peptide, peptidomimetic, small molecule, or other drug, which binds toan RNA-binding protein, modulates, e.g., inhibits, interaction of anRNA-binding protein and an RNA molecule comprising an RNA-bindingprotein recognition element, and/or has a stimulatory or inhibitoryeffect on, for example, RNA-binding protein expression or activity,binding affinity or stability.

For screening assays of the invention, preferably, the “test compound oragent” screened includes molecules that are not known in the art tomodulate activity of an RNA-binding protein and/or expression asdescribed herein. Preferably, a plurality of agents are tested using theinstant methods.

The term “library of test compounds” is intended to refer to a panelcomprising a multiplicity of test compounds.

In one embodiment, the agent or test compound is a compound thatdirectly interacts with the RNA-binding protein or directly interactswith a molecule with which the RNA-binding protein interacts (e.g., acompound that inhibits or stimulates the interaction between theRNA-binding protein and the RNA-binding protein target molecule, e.g.,an RNA molecule comprising an RNA-binding protein recognition element).In another embodiment, the compound is one that indirectly modulatesexpression and/or activity of an RNA-binding protein, e.g., bymodulating the activity of a molecule that is upstream or downstream ofthe RNA-binding protein in a signal transduction pathway involving theRNA-binding protein. Such compounds can be identified using screeningassays that select for such compounds, as described in detail below.

The term “interact” as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a gel shift assay, a fluorescence polarization assay, a yeasttwo hybrid assay, and coimmunoprecipitation. The term interact is alsomeant to include “binding” interactions between molecules. Interactionsmay be protein-protein or protein-nucleic acid in nature.

As used herein, the term “contacting” (e.g., contacting a cell with acompound) is intended to include incubating the compound and the celltogether in vitro (e.g., adding the compound to cells in culture) oradministering the compound to a subject such that the compound and cellsof the subject are contacted in vivo.

As used herein, the term “indicator composition” refers to a compositionthat includes a protein of interest (e.g., an RNA-binding protein), forexample, a cell that naturally expresses the protein, a cell that hasbeen engineered to express the protein by introducing an expressionvector encoding the protein into the cell, or a cell free compositionthat contains the protein (e.g., purified naturally-occurring protein orrecombinantly-engineered protein).

As used herein, the term “cell free composition” refers to an isolatedcomposition which does not contain intact cells. Examples of cell freecompositions include cell extracts and compositions containing isolatedproteins.

In one embodiment, small molecules can be used as test compounds. Theterm “small molecule” is a term of the art and includes molecules thatare less than about 7500, less than about 5000, less than about 1000molecular weight or less than about 500 molecular weight. In oneembodiment, small molecules do not exclusively comprise peptide bonds.In another embodiment, small molecules are not oligomeric. Exemplarysmall molecule compounds which can be screened for activity include, butare not limited to, peptides, peptidomimetics, nucleic acids,carbohydrates, small organic molecules (e.g., Cane et al. 1998. Science282:63), and natural product extract libraries. In another embodiment,the compounds are small, organic non-peptidic compounds. In a furtherembodiment, a small molecule is not biosynthetic. For example, a smallmolecule is preferably not itself the product of transcription ortranslation.

I. RNA Binding Modulatory Compounds

In one embodiment, the RNA binding modulatory compound is a compound offormula I:

wherein

R¹, R², R³ and R⁴ are each independently hydrogen, hydroxyl, alkyl,alkenyl, alkynyl, aryl, amino, sulfonyl, carbonyl, carboxylate, alkoxy,aryloxy, carbonyloxy, acyl or a heterocyclic moiety; or R¹ and R³ and/orR² and R⁴ together with the nitrogen atom to which they are attached arelinked to form a 3-9-membered carbocyclic or heterocyclic ring or5-9-membered aryl ring; and pharmaceutically acceptable salts thereof.

In one embodiment, R¹ and R² are each hydrogen and R³ is aryl, forexample, phenyl or a thiophenyl moiety. In another embodiment, thephenyl is a mono- or di-substituted phenyl, substituted with, forexample, carboxylate, halogen (e.g., chlorine), alkyl (e.g., methyl) oramino (e.g., carbonylamino, for example, alkylcarbonylamino, such asCH₃(CH₂)₃CONH—). In yet another embodiment, the phenyl is selected fromthe group consisting of:

In a further embodiment, the thiophenyl moiety is carbonyl substitutedwith, for example, alkoxy (e.g., ethoxy) and alkyl (e.g., methyl). Inone embodiment, the thiophenyl moiety is

In one embodiment, R⁴ is carbonyl substituted with an aryl moiety, suchas phenyl (e.g., mono-, di- or tri-substituted), for example, phenylsubstituted with halogen (e.g., chlorine, bromine or iodine), alkoxy(e.g., methoxy or ethoxy), nitro, aryl (e.g., phenyl) or alkyl (e.g.,methyl). In another embodiment, the phenyl is selected from the groupconsisting of:

In yet another embodiment, R⁴ is carbonyl substituted with an arylmoiety, such as naphthyl, for example, an alkoxy (e.g., methoxy)substituted naphthyl. In one embodiment, the napthyl moiety is

In one embodiment, R⁴ is carbonyl substituted, for example, with an arylmoiety, such as a heteroaryl moiety, such as, a furanyl orbenzothiophenyl moiety. In another embodiment, the furanyl moiety issubstituted with aryl, for example, phenyl, such as phenyl substitutedwith halogen (e.g., chlorine). In yet another embodiment, the furanylmoiety is

In a further embodiment, the benzothiophenyl moiety is substituted withhalogen (e.g., chlorine). In yet another embodiment, the benzothiophenylmoiety is

In yet another embodiment, R⁴ is carbonyl, for example, carbonylsubstituted with alkyl (e.g., isopropyl).

In one embodiment, R¹ and R³ are each alkyl, for example, arylsubstituted alkyl, such as benzyl.

In a further embodiment, R² is hydrogen and R⁴ is carbonyl, for example,carbonyl substituted with aryl, such as phenyl (e.g., —NO₂ substitutedphenyl, for example,

or a furanyl moiety, such as a furanyl moiety substituted with halogen(e.g., bromine), for example,

In yet another embodiment, R⁴ is a benzo[d]imidazole moiety, forexample, 1-alkyl-1H-benzo[d]imidazole, such as1-propyl-1H-benzo[d]imidazole, 1-butyl-1H-benzo[d]imidazole or1-benzyl-1H-benzo[d]imidazole.

In another embodiment, R¹ is hydrogen and R³ is aryl (e.g., adiphenylquinoxalinyl moiety, for example,

In one embodiment, R² and R⁴ together with the nitrogen atom to whichthey are attached are linked to form a 3-9 membered heterocyclic ring,such as a piperazinyl ring, for example, a piperazinyl ring substitutedwith a carbonyl moiety (e.g., carbonyl substituted with alkoxy, such ascarbonyl substituted with ethoxy). In another embodiment, thepiperazinyl ring is

In a further embodiment, R³ is sulfonyl (e.g., tosylate).

In yet another embodiment, R⁴ is alkyl, for example, aryl substitutedalkyl, such as an oxydibenzene moiety, including, but not limited to,

In another embodiment, the RNA binding modulatory compound of formula Iis a compound of formula Ia:

wherein

R^(1a) and R^(2a) are each independently hydrogen or alkyl; and

R^(3a) and R^(4a) are each independently alkyl or aryl, andpharmaceutically acceptable salts thereof.

In one embodiment, R^(1a) and R^(2a) are each hydrogen and R^(3a) isaryl, such as phenyl or a thiophenyl moiety. In another embodiment, thephenyl is a mono- or di-substituted phenyl, substituted with, forexample, carboxylate, halogen (e.g., chlorine), alkyl (e.g., methyl) oramino (e.g., amino substituted with carbonyl, which may be furthersubstituted with alkyl, for example, CH₃(CH₂)₃CONH—). In a furtherembodiment, the thiophenyl moiety may be substituted with carbonyl(e.g., carbonyl substituted with alkoxy, such as carbonyl substitutedwith ethoxy) and alkyl (e.g., methyl).

In another embodiment, R^(1a) and R^(3a) are each alkyl, for example,alkyl substituted with aryl (e.g., phenyl), such as benzyl.

In yet another embodiment, R^(3a) is selected from the group consistingof:

In one embodiment, R^(4a) is an aryl moiety, for example, phenyl (e.g.,a mono-, di- or tri-substituted), such as phenyl substituted withhalogen (e.g., chlorine, bromine or iodine), alkoxy (e.g., methoxy orethoxy), nitro, aryl (e.g., phenyl) or alkyl (e.g., methyl).

In another embodiment, R^(4a) is aryl, for example, naphthyl, such as anaphthyl substituted with alkoxy (e.g., methoxy).

In yet another embodiment, R^(4a) is an aryl moiety, such as aheteroaryl moiety, for example, a furanyl or benzothiophenyl moiety. Inanother embodiment, the furanyl moiety is substituted with aryl, forexample, phenyl, such as phenyl substituted with halogen (e.g.,chlorine). In a further embodiment, the benzothiophenyl moiety issubstituted with halogen (e.g., chlorine).

In yet another embodiment, R^(4a) is alkyl (e.g., isopropyl).

In one embodiment, R^(4a) is selected from the group consisting of:

In one embodiment, the RNA binding modulatory compound of formula I is acompound of formula Ib:

wherein

R^(1b) and R^(2b) are each hydrogen;

R^(3b) is aryl or sulfonyl; and

R^(4b) is alkyl or aryl; or R^(2b) and R^(4b) together with the nitrogento which they are attached are linked to form a 3-9-memberedheterocyclic or 5-9-membered aryl ring; and pharmaceutically acceptablesalts thereof.

In one embodiment, R^(1b) and R^(2b) are each hydrogen, R^(3b) is asulfonyl (e.g., aryl substituted sulfonyl, for example, tosylate), andR^(4b) is aryl (e.g., an oxydibenzene moiety, for example,

In another embodiment, R^(1b) and R^(2b) are each hydrogen, R^(3b) isaryl (e.g., phenyl) and R^(4b) is aryl, for example, heteroaryl, such asa benzo[d]imidazole moiety (e.g., 1-alkyl-1H-benzo[d]imidazole, forexample, 1-propyl-1H-benzo[d]imidazole, 1-butyl-1H-benzo[d]imidazole or1-benzyl-1H-benzo[d]imidazole).

In a further embodiment, R^(1a) is hydrogen, R^(3b) is aryl, forexample, a diphenylquinoxalinyl moiety

and R^(2b) and R^(4b) together with the nitrogen to which they areattached are linked to form a 3-9 membered heterocyclic ring, forexample, a piperazinyl ring, such as a piperazinyl ring substituted withcarbonyl (e.g., carbonyl substituted with alkoxy, for example, carbonylsubstituted with ethoxy).

In yet another embodiment, the compound of formula I is a compoundselected from the group consisting of:

and pharmaceutically acceptable salts thereof.

In another embodiment, the RNA binding modulatory compound is a compoundof formula II:

wherein

R⁵ and R⁷ are each hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl,amino, sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy, carbonyloxy,acyl or a heterocyclic moiety;

R⁶ is hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfone,carbonyl, carboxylate, alkoxy, aryloxy, carbonyloxy, halogen, acyl,oximyl, hydrazinyl, —NO₂, —CN, a heterocyclic moiety or thioether; andpharmaceutically acceptable salts thereof.

In one embodiment, R⁷ is aryl, for example, phenyl, such as phenylsubstituted with halogen (e.g., bromine); R⁶ is hydrogen and R⁵ isalkyl, such as aminocarbonylalkyl, which may be substituted, forexample, aryl (e.g., phenyl, including, but not limited to, phenylsubstituted with aminocarbonyl. In one embodiment, R⁵ is

In another embodiment, R⁷ is hydrogen; R⁵ is aryl, for example, phenyl(e.g., disubstituted phenyl), for example, phenyl substituted withhydroxyl, halogen (e.g., chlorine), alkyl (e.g., methyl) or amino (e.g.,aryl substituted sulfonylamino, for example, phenyl substitutedsulfonylamino). In one embodiment, R⁵ is selected from the groupconsisting of:

In a further embodiment, R⁶ is —NO₂ or hydrogen.

In yet another embodiment, R⁷ is hydrogen; R⁵ is aryl, for example,heteroaryl, such as an oxazolyl moiety

and R⁶ is halogen (e.g., chlorine).

In a further embodiment, R⁷ is hydrogen; R⁵ is alkyl, for example,heteroaryl substituted alkyl in which the heteroaryl may be abenzo[d]imidazole moiety, (e.g., a 1[H]-benzo[d]imidazole) and R⁶ isalkyl (e.g., methyl) or halogen (e.g., chlorine).

In yet another embodiment, R⁷ is hydrogen; R⁵ is heteroaryl, forexample, isoxazolyl, thiazolyl, thiadiazolyl or pyrimidinyl such as

R⁶ is amino, for example, amino substituted with sulfonyl, for example,sulfonyl substituted with aryl in which the aryl may be substituted withhalogen (e.g., bromine); aryl, for example, phenyl, such astrisubstituted phenyl substituted with halogen (e.g., chlorine) andhydroxyl; heteroaryl, for example, thiazolyl, which may be substitutedwith aryl, such as phenyl, for example, phenyl substituted withhydroxyl, alkyl (e.g., methyl) or halogen (e.g., fluorine). In anotherembodiment, R⁶ is selected from the group consisting of:

In yet another embodiment, R⁷ is hydrogen; R⁵ is aryl, for example,heteroaryl (e.g., isoxazolyl, thiazolyl or thiadiazolyl, such as

and R⁶ is amino, for example, carbonylamino, such as carbonylaminosubstituted with alkyl. In one embodiment, the alkyl is C₁-C₆ alkyl thatmay be substituted with thiol, for example heteroaryl (e.g.,benzothiazolyl or triazinyl) substituted thiol; aryloxy, for example,phenoxy, such as di- or trisubstituted phenoxy substituted with alkyl(e.g., methyl) or halogen (e.g., bromine); a heterocyclic moiety (e.g.,quinazolinonyl) or aryl, for example, phenyl, such as phenyl substitutedwith alkyl (e.g., methyl).

In yet another embodiment, R⁷ is hydrogen; R⁵ is aryl, for exampleheteroaryl (e.g., isoxazolyl, thiazolyl or thiadiazolyl, such as

and R⁶ is amino, for example carbonylamino, such as carbonylaminosubstituted with aryl, for example, phenyl (e.g., mono- ordi-substituted phenyl), such as phenyl substituted with alkyl (e.g.,t-butyl), alkoxy (e.g., methoxy or n-butoxy) or halogen (e.g., chlorineor bromine).

In another embodiment, R⁷ is hydrogen; R⁵ is aryl, for exampleheteroaryl (e.g., isoxazolyl, thiazolyl or thiadiazolyl, such as

and R⁶ is amino, for example, carbonylamino, such as carbonylaminosubstituted with a heterocyclic moiety (e.g., chromene orpyrrolidinone).

In a further embodiment, R⁶ is selected from the group consisting of:

In one embodiment, the RNA binding modulatory compound of formula II isa compound of formula IIa:

wherein

R^(5a) is alkyl or aryl;

R^(6a) is —NO₂, alkyl, halogen or hydrogen; and

R^(7a) is hydrogen or aryl, and pharmaceutically acceptable saltsthereof.

In one embodiment, R^(7a) is aryl, for example, phenyl, such as phenylsubstituted with halogen (e.g., bromine); R^(6a) is hydrogen and R^(5a)is alkyl, such as, aminocarbonylalkyl, which may be substituted, forexample, with aryl (e.g., phenyl, including, but not limited to, phenylsubstituted with aminocarbonyl). In one embodiment, R⁵ is

In another embodiment, R^(7a) is hydrogen; R^(5a) is aryl, for example,phenyl (e.g., disubstituted phenyl), for example, phenyl substitutedwith hydroxyl, halogen (e.g., chlorine), alkyl (e.g., methyl) or amino(e.g., aryl substituted sulfonylamino, for example, phenyl substitutedsulfonylamino). In one embodiment, R^(5a) is selected from the groupconsisting of:

In a further embodiment, R^(6a) is —NO₂ or hydrogen.

In yet another embodiment, R^(7a) is hydrogen; R^(5a) is aryl, forexample, heteroaryl, such as an oxazolyl moiety

and R^(6a) is halogen (e.g., chlorine).

In a further embodiment, R^(7a) is hydrogen; R^(5a) is alkyl, forexample, heteroaryl substituted alkyl in which the heteroaryl may be abenzo[d]imidazole moiety, (e.g., a 1[H]-benzo[d]imidazole) and R^(6a) isalkyl (e.g., methyl) or halogen (e.g., chlorine).

In another embodiment, the RNA binding modulatory compound of formula IIis a compound of formula IIb:

wherein

R^(5b) is aryl; and

R^(6b) is sulfonyl or aryl, and pharmaceutically acceptable saltsthereof.

In yet another embodiment, R^(5b) is aryl, such as heteroaryl, forexample, isoxazolyl, thiazolyl, thiadiazolyl or pyrimidinyl, such as

R^(6b) is sulfonyl, for example, sulfonyl substituted with aryl in whichthe aryl may be substituted with halogen (e.g., bromine); or aryl, forexample, phenyl, such as trisubstituted phenyl substituted with halogen(e.g., chlorine) and hydroxyl; heteroaryl, for example, thiazolyl, whichmay be substituted with aryl such as phenyl, for example, substitutedwith hydroxyl, alkyl (e.g., methyl) or halogen (e.g., fluorine). Inanother embodiment, R^(6b) is selected from the group consisting of:

In one embodiment, the RNA binding modulatory compound of formula II isa compound of formula IIc:

wherein

R^(5c) is aryl; and

R^(6c) is alkyl, aryl or a heterocyclic moiety, and pharmaceuticallyacceptable salts thereof.

In yet another embodiment, R^(5c) is aryl, for example heteroaryl (e.g.,isoxazolyl, thiazolyl or thiadiazolyl, such as

and R^(6c) is alkyl, for example, C₁-C₆ alkyl that may be substitutedwith thiol, such as heteroaryl (e.g., benzothiazolyl or triazinyl)substituted thiol; aryloxy, for example, phenoxy, such as di- ortrisubstituted phenoxy substituted with alkyl (e.g., methyl) or halogen(e.g., bromine); a heterocyclic moiety (e.g., quinazolinonyl) or aryl,for example, phenyl, such as phenyl substituted with alkyl (e.g.,methyl).

In yet another embodiment, R^(5c) is aryl, for example heteroaryl (e.g.,isoxazolyl, thiazolyl or thiadiazolyl, such as

and R^(6c) is aryl for example, phenyl (e.g., mono- or di-substitutedphenyl), such as phenyl substituted with alkyl (e.g., t-butyl), alkoxy(e.g., methoxy or n-butoxy) or halogen (e.g., chlorine or bromine).

In another embodiment, R^(5c) is aryl, for example heteroaryl (e.g.,isoxazolyl, thiazolyl or thiadiazolyl, such as

and R^(6c) is a heterocyclic moiety (e.g., chromenyl or pyrrolidinonyl).

In a further embodiment, R^(6c) is selected from the group consistingof:

In one embodiment, the compound of formula II is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.

In yet another embodiment, the RNA binding modulatory compound is acompound of formula III:

wherein

R⁸, R⁹, R¹⁰ and R¹¹ are each independently hydrogen, hydroxyl, alkyl,alkenyl, alkynyl, aryl, amino, sulfone, carbonyl, carboxylate, alkoxy,aryloxy, carbonyloxy, halogen, acyl, oximyl, hydrazinyl, —NO₂, —CN, aheterocyclic moiety or thioether;

R¹² is —NR¹⁴SO₂R¹⁵;

R¹³ is —NR¹⁶SO₂R¹⁷;

R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are each independently hydrogen, hydroxyl, alkyl,alkenyl, alkynyl, aryl, amino, sulfonyl, carbonyl, carboxylate, alkoxy,aryloxy, carbonyloxy, acyl, halogen or a heterocyclic moiety; or R¹⁴ andR¹⁵ and/or R¹⁶ and R¹⁷ together with the nitrogen atom to which they areattached are linked to form a 3-9-membered heterocyclic ring or 5-9 arylring; and pharmaceutically acceptable salts thereof.

In another embodiment, R¹⁴ and R¹⁶ are each hydrogen; R¹⁵ and R¹⁷ areeach aryl, for example, phenyl, such as unsubstituted phenyl; R⁸, R⁹,and R¹¹ are each hydrogen and R¹⁰ is —NO₂.

In another embodiment, R¹⁴ and R¹⁶ are each hydrogen; R⁸, R¹⁰ and R¹¹are each hydrogen; R⁹ is alkyl (e.g., methyl) or hydrogen; and R¹⁵ andR¹⁷ are each aryl, for example, phenyl, such as phenyl substituted withalkoxy (e.g., methoxy) or halogen (e.g., fluorine). In one embodiment,the phenyl is

In yet another embodiment, R¹⁴ and R¹⁶ are each hydrogen; R¹⁵ and R¹⁷are each alkyl (e.g., ethyl) and R⁸, R⁹, R¹⁰ and R¹¹ are each hydrogen.

In one embodiment, the compound is selected from the group consistingof:

and pharmaceutically acceptable salts thereof.

In a further embodiment, the RNA binding modulatory compound is acompound of formula IV:

wherein

R¹⁸ and R¹⁹ are each independently hydrogen, hydroxyl, alkyl, alkenyl,alkynyl, aryl, amino, sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy,carbonyloxy, acyl or a heterocyclic moiety; or R¹ and R³ and/or R² andR⁴ together with the nitrogen atom to which they are attached are linkedto form a 3-9-membered heterocyclic or 5-9-membered heteroaryl ring; and

R²⁰ is hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino,sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy, carbonyloxy, acyl,halogen or a heterocyclic moiety; and pharmaceutically acceptable saltsthereof.

In one embodiment, R¹⁸ is hydrogen; R²⁰ is aryl, for example,heteroaryl, such as thiophenyl, benzothiazolyl or phthalazinyl

and R¹⁹ is acyl (—COCH₃), alkyl, for example, alkyl substituted with aheterocyclic moiety (e.g., tetrahydrofuranyl), such as

or aryl, such as naphthyl, for example, naphthyl substituted withhalogen (e.g., chlorine) and hydroxyl

In one embodiment, R¹⁸ is hydrogen; R²⁰ is aryl, for example, phenyl(e.g., mono- or disubstituted phenyl), for example, phenyl substitutedwith —NO₂, alkyl (e.g., methyl) or aminocarbonyl (e.g., —CONH₂), such as

and R¹⁹ is aryl, for example, naphthyl, such naphthyl substituted withhalogen (e.g., chlorine or bromine) and hydroxyl

or phenyl, for example, phenyl substituted with alkoxy (e.g., ethoxy).

In yet another embodiment, the RNA binding modulatory compound offormula III is selected from the group consisting of:

and pharmaceutically acceptable salt thereof.

In one embodiment, the RNA binding modulatory compound is a compound offormula V:

wherein

R²¹, R²², R²³ and R²⁴ are each independently hydrogen, hydroxyl, alkyl,alkenyl, alkynyl, aryl, amino, sulfone, carbonyl, carboxylate,carbonyloxy, alkoxy, aryloxy, halogen, acyl, oximyl, hydrazinyl, —NO₂,—CN, a heterocyclic moiety or thioether;

R²⁵ is hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino,sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy, carbonyloxy, acyl or aheterocyclic moiety; and

R²⁶ is hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino,sulfonyl, carbonyl, carboxylate, alkoxy, aryloxy, carbonyloxy, acyl or aheterocyclic moiety; and pharmaceutically acceptable salts thereof.

In another embodiment, R²¹, R²⁵ and R²⁶ are hydrogen; and R²³ ishydrogen and R²² and R²⁴ are each halogen (e.g., chlorine).

In yet another embodiment, R²¹, R²², R²⁴, R²⁵ and R²⁶ are hydrogen; andR²³ is carboxy, for example, carboxy substituted with aryl, such asnaphthyl or phenyl (e.g., phenyl substituted with alkoxycarbonyl, forexample, methoxycarbonyl or ethoxycarbonyl). In one embodiment, thecarboxy is

In yet another embodiment, R²¹, R²², R²⁴, R²⁵ and R²⁶ are hydrogen; andR²³ is carbonyloxy, such as carbonyloxy substituted with aryl (e.g.,phenyl, including disubstituted phenyl, which may be substituted withhalogen (e.g., chlorine)). In one embodiment, the carbonyloxy is

In one embodiment, the compound of formula V is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.

In another embodiment, the RNA binding modulatory compound is a compoundof formula VI:

wherein

R²⁷, R²⁸, R²⁹, R³⁰ and R³¹ are each independently hydrogen, hydroxyl,alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl, carbonyl, carboxylate,carbonyloxy, alkoxy, aryloxy, halogen, acyl, oximyl, hydrazinyl, —NO₂,—CN, a heterocyclic moiety or thioether; or R²⁷ and R²⁸ or R²⁸ and R²⁹or R²⁹ and R³⁰ or R³⁰ and R³¹ together with the carbon atoms to whichthey are attached are linked to form a 4-9 carbocyclic, heterocyclic oraryl ring; and pharmaceutically acceptable salts thereof.

In one embodiment, R²⁷ and R²⁸ are linked to form an aryl moiety (e.g.,phenyl); R³⁰ is hydrogen; R³¹ is aryl, for example, furanyl

and R²⁹ is carbonyl, for example, carbonyl substituted with amino, whichmay in turn be substituted with aryl, for example, phenyl, such asphenyl substituted with halogen (e.g., chlorine). In one embodiment, R²⁹is

In another embodiment, R²⁷, R²⁸ and R²⁹ are each hydrogen and R³⁰ andR³¹ are linked to form an aryl moiety, for example, phenyl (e.g.,tri-substituted phenyl), for example, phenyl substituted with —NO₂,hydroxyl and alkyl, which may be substituted with a heterocyclic moiety,amino or aryl. In one embodiment, the heterocyclic moiety is morpholineor pyrrolidine, which may be substituted with alkyl (e.g., methyl). Inone embodiment, the amino is substituted with carbonyl, which may befurther substituted with alkyl (e.g., methyl, ethyl, n-propyl orn-butyl). In one embodiment, the aryl is furanyl or phenyl, for example,phenyl substituted with halogen (e.g., chlorine), hydroxyl or alkoxy(e.g., methoxy or alkoxy). In one embodiment, R³⁰ and R³¹ are linked toform a ring selected from the group consisting of

In yet another embodiment, R²⁸, R²⁹ and R³⁰ are each hydrogen and R²⁷and R³¹ are each substituted with carbonyl, for example, carbonylsubstituted with amino, which may be substituted with aryl, such asthiadiazolyl (e.g., thiadiazolyl substituted with alkyl, for example,methyl or n-propyl) or benzothiazolyl. In one embodiment, R²⁷ and R³¹are

In a further embodiment, R²⁷, R²⁹ and R³⁰ are each hydrogen and R²⁸ andR³¹ are each carbonyl, for example, carbonyl substituted with amino,which may be further substituted with alkyl (e.g., alkyl substitutedwith aryl, for example, phenyl, such as phenyl substituted with alkoxy(e.g., methoxy). In one embodiment, R²⁸ and R³¹ are each

In one embodiment, the RNA binding modulatory compound of formula VI isa compound of formula VIa:

wherein

R^(30a) and R^(31a) are each —NO₂ or alkyl; and pharmaceuticallyacceptable salts thereof.

In one embodiment, R^(31a) is —NO₂ and R^(30a) is alkyl, which may besubstituted with a heterocyclic moiety (e.g., morpholine).

In another embodiment, R^(30a) is —NO₂ and R^(31a) is alkyl, forexample, alkyl substituted with a heterocyclic moiety, amino or aryl. Inone embodiment, the heterocyclic moiety is pyrrolidine, which may besubstituted with alkyl (e.g., methyl). In one embodiment, the amino issubstituted with carbonyl, which may be further substituted with alkyl(e.g., methyl, ethyl, n-propyl or n-butyl). In one embodiment, the arylis furanyl or phenyl, for example, phenyl substituted with halogen(e.g., chlorine), hydroxyl or alkoxy (e.g., methoxy or alkoxy). In oneembodiment, R^(31a) is selected from the group consisting of

In yet another embodiment, the RNA binding modulatory compound offormula VI is a compound of formula VIb:

wherein

R^(27b) and R^(31b) are each carbonyl; and pharmaceutically acceptablesalts thereof.

In one embodiment, the carbonyl is substituted with amino, which may besubstituted with aryl, such as thiadiazolyl (e.g., thiadiazolylsubstituted with alkyl, for example, methyl or n-propyl) orbenzothiazolyl. In one embodiment, the carbonyl is substituted with

In another embodiment, the compound of formula VI is selected from thegroup consisting of

and pharmaceutically acceptable salts thereof.

In yet another embodiment, the RNA binding modulatory compound is acompound of formula VII:

wherein

U is NR³², O or S;

W is N or CR³³ when q is a double bond, or W is NR⁴⁴, CR⁴⁵R⁴⁶ or C=Dwhen q is a single bond;

X is N or CR³⁴ when q is a double bond, or X is NR⁴⁷, CR⁴⁸R⁴⁹ or C=Ewhen q is a single bond;

Y is NR³⁵, CR³⁶R³⁷ or C=G when p is a single bond, or Y is N or CR³⁸when p is a double bond;

Z is NR³⁹, CR⁴⁰R⁴¹ or C=A when p is a single bond, or Z is N or CR⁴²when p is a double bond;

p and q are each independently a single or double bond;

R³², R³⁵, R³⁹, R⁴³, R⁴⁴, R⁴⁷, R⁵², R⁵⁵ and R⁵⁸ are each independentlyhydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl,carbonyl, carboxylate, alkoxy, aryloxy, carbonyloxy, acyl or aheterocyclic moiety;

R³³, R³⁴, R³⁶, R³⁷, R³⁸, R⁴⁰, R⁴¹, R⁴², R⁴⁵, R⁴⁶, R⁴⁸, R⁴⁹, R⁵⁰, R⁵¹,R⁵³, R⁵⁴, R⁵⁶, R⁵⁷, R⁵⁹ and R⁶⁰ are each independently hydrogen,hydroxyl, alkyl, alkenyl, alkynyl, aryl, amino, sulfonyl, carbonyl,carboxylate, alkoxy, aryloxy, carbonyloxy, halogen, acyl, oximyl,hydrazinyl, —NO₂, —CN, a heterocyclic moiety or thioether; or

R³³ and R³⁴ together with the atoms to which they are attached arelinked to form a 4-12 membered carbocyclic, aryl or heterocyclic ring;or

R³⁵, R³⁶ or R³⁸ and R³⁹, R⁴⁰ or R⁴¹ together with the atoms to whichthey are attached are linked to form a 4-12 membered carbocyclic, arylor heterocyclic ring; or

R³² and R³³ together with the atoms to which they are attached arelinked to form a 4-12 membered carbocyclic, aryl or heterocyclic ring;and

A is O, S, NR⁴³ or CR⁵⁰R⁵¹;

D is O, S, NR⁵² or CR⁵³R⁵⁴;

E is O, S, NR⁵⁵ or CR⁵⁶R⁵⁷;

G is O, S, NR⁵⁸ or CR⁵⁹R⁶⁰; and pharmaceutically acceptable saltsthereof.

In one embodiment, p and q are each a double bond; U is O; W is CR³³ andZ is CR⁴²; X is N; Y is N; R⁴² is thiol (e.g., —SH) or aryl, forexample, phenyl, such as phenyl substituted with halogen (e.g., bromine)or quinoline, for example, quinoline substituted with aryl (e.g.,phenyl); R³³ is thiol, for example, thiol substituted with aryl (e.g.,phenyl, which may be substituted with —NO₂ and carboxylate) or aryl. Inone embodiment, R³³ is

and R⁴² is

Alternatively, R³³ is

In another embodiment, Y is CR³⁸; R³⁸ and R⁴² are each aryl (e.g.,phenyl) and R³³ is amino, for example, amino substituted with carbonyl,which may be substituted with aryl (e.g., phenyl, for example phenylsubstituted with alkyl, such as methyl) In one embodiment, R³³ is

In yet another embodiment, p and q are each a double bond; U is S; W isCR³³ and Z is CR⁴²; X is N; Y is N; R³³ is amino or thiol. In oneembodiment, R³³ is amino, for example, amino substituted with carbonylor oximyl. In another embodiment, the oximyl is an aryl substitutedoximyl, such as phenyl substituted oximyl in which the phenyl may befurther substituted with —NO₂. In yet another embodioxy, such as ethoxy)or aryl (e.g., heteroaryl, for example, triazole). Alternatively, R³³ isthiol, for example, thiol substituted with alkyl (e.g., aryl substitutedalkyl, such as phenyl substituted alkyl, which may be furthersubstituted with halogen, such as chlorine). In one embodiment, R³³ is

In another embodiment, R⁴² is alkyl, for example, t-butyl or alkylsubstituted with aryl (e.g., phenyl, for example phenyl substituted withhalogen, such as chlorine) or carbonyl (e.g., alkoxy substitutedcarbonyl, for example, ethoxyl substituted carbonyl). Alternatively R⁴²is aryl (e.g., heteroaryl, such as pyridine) or thiol (e.g., —SH). Inanother embodiment, R⁴² is

In yet another embodiment, p and q are each a double bond; U is S; W isCR³³ and Z is CR⁴²; X is N; Y is CR³⁸; R⁴² is hydrogen; R³⁸ is aryl, forexample, phenyl, such as phenyl substituted with hydroxyl

and R³³ is amino, for example, amino substituted with aryl, such as,naphthyl

In another embodiment, p and q are each a double bond; U is S; W is CR³³and Z is CR⁴²; X is CR³⁴; Y is CR³⁸; R³³ and R³⁴ are each alkyl (e.g.,methyl); R³⁸ is carbonyl, for example, carbonyl substituted with amino(e.g., —NH₂); R⁴² is amino, for example, amino substituted withcarbonyl, such as aryl substituted carbonyl (e.g., pyrazine substitutedcarbonyl, for example,

and R³³ is amino (e.g., amino substituted with carbonyl, such ascarbonyl substituted with cyclohexenyl, for example,

In another embodiment, R³⁴ is carbonyl, for example, carbonylsubstituted with alkoxy (e.g., methoxy); R⁴² is alkyl (e.g., ethyl); andR³⁸ is aryl, for example, phenyl, such as phenyl substituted withhalogen (e.g., chlorine). In one embodiment, R³⁸ is

In yet another embodiment, R³⁸ and R⁴² are linked to join a ring (e.g.,a 6-membered or 12-membered carbocyclic ring), for example,

In one embodiment, R³³ is amino, for example, amino substituted withcarbonyl (e.g., alkyl substituted carbonyl such as alkyl substitutedwith carboxylate). In one embodiment, R³³ is

In another embodiment, R³⁴ is carbonyl, for example, alkoxy (e.g.,methoxy) substituted carbonyl.

In another embodiment, R³³ and R³⁴ are linked to form a 6-memberedheterocyclic ring, for example,

In yet another embodiment, p and q are each a double bond; U is NR³²; Xis N; Y is N; W is CR³³ and Z is CR⁴²; R⁴² is thiol (e.g., —SH); R³² ishydrogen, alkyl (e.g., methy or alkyl substituted with aryl, forexample, phenyl, or alkenyl) or aryl, for example, unsubstituted phenylor phenyl substituted with alkyl (e.g., methyl) or halogen (e.g.,fluorine). In one embodiment, R³² is

In a further embodiment, R³³ is alkyl, for example, alkyl substitutedwith aryloxy, for example, naphthyloxy or phenoxy, such as halogen(e.g., bromine) substituted phenoxy, alkyl (e.g., methyl) substitutedphenoxy or alkoxy (e.g., methoxy) substituted phenoxy. In anotherembodiment R³³ is a thiol substituted alkyl (e.g., arylalkylthioalkyl,such as phenyl substituted alkylthioalkyl in which the phenyl may befurther substituted with halogen, for example chlorine); a heterocyclicsubstituted alkyl (e.g., alkyl substituted with

aryl substituted alkyl, for example, phenyl substituted alkyl (e.g.,unsubstituted phenyl or phenyl substituted with halogen, such aschlorine, or phenyl substituted with alkoxy, such as isopropoxy).Alternatively, R³³ is aryl, for example, thiophenyl or phenyl, forexample, phenyl substituted with hydroxyl or alkoxy (e.g., methoxy). Inanother embodiment, R³³ is selected from the group consisting of

In another embodiment, R³² and R³³ are linked to form a 6-memberedheterocyclic ring

In another embodiment, U is NR³²; X is N; Y is N; W is CR³³; Z is CR⁴²;R³² is hydrogen; R³³ is hydrogen; R⁴² is amino, for example, aminosubstituted with carbonyl (e.g., carbonyl substituted with aryl, such asphenyl, which may be substituted with hydroxyl). In yet anotherembodiment, R⁴² is a heterocyclic moiety

In another embodiment, U is NR³²; W is CR³³, X is CR³⁴, Y is CR³⁵; Z isN; R³² is hydrogen or alkyl (e.g., alkyl substituted with carboxylate,for example, —CH₂CH₂COOH); R³³ is hydrogen or alkyl (e.g., methyl); R³⁴is alkenyl (e.g., alkenyl substituted with a heterocyclic moiety, forexample,

or aryl (e.g., phenyl or benzothiazolyl); R³⁸ is aryl, for example,phenyl, such as phenyl substituted with hydroxyl, alkoxy (e.g.,methoxy), alkyl (e.g., ethyl) or a heterocyclic moiety. In oneembodiment, R³⁸ is

In another embodiment, p and q are each a double bond; U is NR³²; W isCR³³, X is N, Y is CR³⁸; Z is N; R³² is aryl (e.g., phenyl); R³³ is aheterocyclic moiety

R³⁸ is amino, such as, amino substituted with carbonyl (e.g., carbonylsubstituted with aryl, for example, phenyl, such as phenyl substitutedwith alkoxy (e.g., methoxy), alkyl (e.g., methyl)) or sulfonyl (e.g.,sulfonyl substituted with aryl, for example, phenyl, which may besubstituted with alkyl (e.g., methyl)). In one embodiment, R³⁸ is

In another embodiment, R³² and R³³ are linked to form a 5 or 6-memberedheterocyclic ring, for example

In another embodiment, R³⁸ is amino (e.g., amino substituted withcarbonyl, for example, aryl substituted carbonyl, such as phenylsubstituted carbonyl, including —NO₂ substituted phenyl) or aryl (e.g.,phenyl, for example, phenyl substituted with carbonyloxy, such as alkylsubstituted carbonyloxy, including, but not limited to methylsubstituted carbonyloxy). In one embodiment, R³⁸ is

In another embodiment, p and q are each a double bond; U is NR³²; W isN; X is N; Y is N; Z is CR⁴²; R³² is hydrogen; R⁴² is amino, forexample, amino substituted with carbonyl (e.g., aryl substitutedcarbonyl, for example, heteroaryl substituted carbonyl, such asthiophenyl substituted carbonyl

In yet another embodiment, U is NR³²; q is a double bond and p is asingle bond; W is CR³³; X is N; Y is NR³⁵ and Z is C=A; A is S; R³⁵ ishydrogen; R³² is hydrogen, alkyl (e.g., methyl, ethyl or arylsubstituted alkyl, for example, phenyl substituted alkyl) or aryl (e.g.,phenyl, such as phenyl substituted with alkyl, such as methyl or ethyl,for example,

R³³ is aryl, such as furanyl or phenyl, for example, phenyl substitutedwith hydroxyl, halogen (e.g., bromine or chlorine) and alkoxy (e.g.,methoxy). In one embodiment, R³³ is

In one embodiment, U is NR³²; q is a double bond and p is a single bond;W is N; X is CR³⁴; Y is C=G; Z is C=A; A is O; R³² is aryl, for example,phenyl substituted with sulfonyl (e.g., amino substituted sulfonyl),carboxylate or halogen (e.g., chlorine), such as

R³⁴ is alkyl (e.g., methyl); G is CR⁵⁹R⁶⁰, R⁵⁹ is hydrogen and R⁶⁰ isaryl, for example, phenyl, such as phenyl substituted with alkoxy (e.g.,methoxy or ethoxy) or carbonyloxy, for example, carbonyloxy substitutedwith aryl (e.g., heteroaryl, for example, thiophenyl or furanyl). In oneembodiment, R⁶⁰ is

In yet another embodiment, U is NR³²; p and q are each a single bond; Wis NR⁴⁴, X is C=E, Y is C=G; Z is C=A; R⁴⁴ is hydrogen; A and E are O;R³² is aryl, for example, phenyl, such as unsubstituted phenyl or phenylsubstituted with halogen (e.g., chlorine or fluorine) or carbonyl, forexample, carbonyl substituted with alkoxy (e.g., ethoxy), for example,

G is CR⁵⁹R⁶⁰; R⁵⁹ is hydrogen; R⁶⁰ is aryl, for example, phenyl, such asphenyl substituted with alkoxy (e.g., methoxy) or carbonyloxy (e.g.,carbonyloxy substituted with aryl, for example, heteroaryl such asthiophenyl or furanyl). In one embodiment, R⁶⁰ is

In one embodiment, the RNA binding modulatory compound of formula VII isa compound of formula VIIa:

wherein

U^(a) is O or S;

R^(33a) is amino, thiol or aryl; and

R^(42a) is aryl, thiol, or alkyl; and pharmaceutically acceptable saltsthereof.

In one embodiment, U^(a) is S; R^(33a) is amino, for example, aminosubstituted with carbonyl or oximyl. In another embodiment, the oximylis aryl substituted oximyl, such as phenyl substituted oximyl in whichthe phenyl may be further substituted with —NO₂. In yet anotherembodiment, the carbonyl may be substituted with carbonyl (e.g.,carbonyl substituted with alkoxy, such as ethoxy) or aryl (e.g.,heteroaryl, for example, triazole). Alternatively, R^(33a) is thiol, forexample, thiol substituted with alkyl (e.g., aryl substituted alkyl,such as phenyl substituted alkyl, which may be further substituted withhalogen such as chlorine). In one embodiment, R^(33a) is

In another embodiment, R^(42a) is alkyl, for example, t-butyl or alkylsubstituted with aryl (e.g., phenyl, for example, phenyl substitutedwith halogen, such as chlorine) or carbonyl (e.g., alkoxy substitutedcarbonyl, for example, ethoxy substituted carbonyl). AlternativelyR^(42a) is aryl (e.g., heteroaryl, such as pyridine) or thiol (e.g.,—SH). In another embodiment, R⁴² is

In another embodiment, U^(a) is O; R^(42a) is thiol (e.g., —SH) or aryl,for example, phenyl, such as phenyl substituted with halogen (e.g.,bromine) or quinoline, for example, quinoline substituted with aryl(e.g., phenyl); R^(33a) is thiol, for example, thiol substituted witharyl (e.g., phenyl, which may be substituted with —NO₂ and carboxylate)or aryl. In one embodiment, R^(33a) is

and R^(42a) is

Alternatively, R^(33a) is

In another embodiment, the RNA binding modulatory compound of formulaVII is a compound of formula VIIb:

wherein

R^(32b) is hydrogen, aryl or alkyl; and

R^(33b) is alkyl or aryl; or R^(32b) and R^(33b) together with the atomsto which they are attached are linked to form a 6-membered heterocyclicring; and pharmaceutically acceptable salts thereof.

In one embodiment, R^(32b) is hydrogen, alkyl (e.g., methy or alkylsubstituted with aryl, for example, phenyl, or alkenyl) or aryl, forexample, unsubstituted phenyl or phenyl substituted with alkyl (e.g.,methyl) or halogen (e.g., fluorine). In one embodiment, R^(32b) is

In a further embodiment, R^(33b) is alkyl, for example, alkylsubstituted with aryloxy, for example, naphthyloxy or phenoxy, such ashalogen (e.g., bromine) substituted phenoxy, alkyl (e.g., methyl)substituted phenoxy or alkoxy (e.g., methoxy) substituted phenoxy. Inanother embodiment R^(33b) is a thiol substituted alkyl (e.g.,arylalkylthioalkyl, such as phenyl substituted alkylthioalkyl in whichthe phenyl may be further substituted with halogen, for examplechlorine); a heterocyclic substituted alkyl (e.g., alkyl substitutedwith

aryl substituted alkyl, for example, phenyl substituted alkyl (e.g.,unsubstituted phenyl or phenyl substituted with halogen, such aschlorine, or phenyl substituted with alkoxy, such as isopropoxy).Alternatively, R^(33b) is aryl, for example, thiophenyl or phenyl, forexample, phenyl substituted with hydroxyl or alkoxy (e.g., methoxy). Inanother embodiment, R^(33b) is selected from the group consisting of

In another embodiment, R^(32b) and R^(33b) are linked to form a6-membered heterocyclic ring

In yet another embodiment, the RNA binding modulatory compound offormula VII is a compound of formula VIIIc:

wherein

R^(38c) is amino; and pharmaceutically acceptable salts thereof.

In one embodiment, R^(38c) is substituted with carbonyl (e.g., carbonylsubstituted with aryl, for example, phenyl, such as phenyl substitutedwith alkoxy, e.g., methoxy, or alkyl, for example, methyl) or sulfonyl(e.g., sulfonyl substituted with aryl, for example, phenyl, which may besubstituted with alkyl, for example, methyl). In one embodiment, R^(38c)is

In a further embodiment, the RNA binding modulatory compound of formulaVII is a compound of formula VIId:

wherein

R^(33d) is alkyl; and

R^(38d) is aryl or amino; and pharmaceutically acceptable salts thereof.

In one embodiment, R^(33d) is n-butyl. In another embodiment, R^(33d) issubstituted alkyl, for example, carbonyl substituted alkyl, such asaminocarbonyl substituted alkyl

In one embodiment, R^(38d) is amino (e.g., amino substituted withcarbonyl, for example, aryl substituted carbonyl, such as phenylsubstituted carbonyl, including —NO₂ substituted phenyl) or aryl (e.g.,phenyl, for example, phenyl substituted with carbonyloxy, such as alkylsubstituted carbonyloxy, including, but not limited to methylsubstituted carbonyloxy). In one embodiment, R^(38d) is

In a further embodiment, the RNA binding modulatory compound of formulaVII is a compound of formula VIIe:

wherein

R^(32e) is hydrogen, alkyl or aryl; and

R^(33e) is aryl; and pharmaceutically acceptable salts thereof.

In one embodiment, R^(32e) is hydrogen, alkyl (e.g., methyl, ethyl oraryl substituted alkyl, for example, phenyl substituted alkyl) or aryl(e.g., phenyl, for example, phenyl substituted with alkyl, such asmethyl or ethyl, for example,

R^(33e) is aryl such as furanyl or phenyl, for example, phenylsubstituted with hydroxyl, halogen (e.g., bromine or chlorine) andalkoxy (e.g., methoxy). In one embodiment, R^(33e) is

In one embodiment, the RNA binding modulatory compound of formula VII isa compound of formula VIIf:

wherein

NR^(32f) is aryl;

q^(f) is a single or double bond;

W^(f) is NH when q^(f) is a single bond; or N when q^(f) is a doublebond;

X^(f) is C═O is when q^(f) is a single bond; or CR^(34f) when q^(f) is adouble bond;

R^(34f) is alkyl;

R^(60f*) is hydrogen, alkoxy or carbonyloxy; and

R^(60f**) is hydrogen or carbonyloxy; and pharmaceutically acceptablesalts thereof.

In one embodiment, q^(f) is a double bond, X^(f) is CR^(34f); W^(f) isN; R^(34f) is alkyl (e.g., methyl); R^(32f) is phenyl substituted withsulfonyl (e.g., amino substituted sulfonyl), carboxylate or halogen(e.g., chlorine), such as

R^(60f*) is hydrogen or alkoxy (e.g., methoxy or ethoxy) and R^(60f**)is carbonyloxy, for example, aryl substituted carbonyloxy, such asfuranyl or thiophenyl substituted carbonyloxy.

In another embodiment, q^(f) is a single bond, W^(f) is NH; X^(f) isC═O; R^(32f) is phenyl, such as unsubstituted phenyl or phenylsubstituted with halogen (e.g., chlorine or fluorine) or carbonyl, forexample, carbonyl substituted with alkoxy (e.g., ethoxy), for example,

R^(60f*) is hydrogen, alkoxy (e.g., methoxy) or carbonyloxy, forexample, aryl substituted carbonyloxy, such as furanyl or thiophenylsubstituted carbonyloxy; and R^(60f**) is hydrogen or carbonyloxy, forexample, aryl substituted carbonyloxy, such as furanyl or thiophenylsubstituted carbonyloxy.

In another embodiment, the RNA binding modulatory compound of formulaVII is a compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

In yet another embodiment, the RNA binding modulatory compound is acompound of Table 1:

TABLE 1

and pharmaceutically acceptable salts thereof.

In one embodiment, the compound is not abamectin, praziquantel,albendazole, diethylcarbamazine, mebendazole, niclosamide, ivermectin,suramin, thiabendazole, pyrantel pamoate, levamisole, triclabendazole,flubendazole, fenbendazole, octadepsipeptide, oxamniquine, metrifonate,bithionol, niridazole, stibophen, ciclobendazole, oxantel, pyrvinium,bephenium, desapidin or dichlorophen.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. The term alkyl further includes alkyl groupsthat may include oxygen, nitrogen, sulfur or phosphorous atoms replacingone or more carbons of the hydrocarbon backbone. In certain embodiments,a straight chain or branched chain alkyl has 6 or fewer carbon atoms inits backbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain).Likewise, cycloalkyls may have from 3-8 carbon atoms in their ringstructure. The term “C₁-C₆” includes alkyl groups containing 1 to 6carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls,” the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogen, hydroxyl, aryl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonate, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyls can befurther substituted, e.g., with the substituents described above. An“alkylaryl” or an “arylalkyl” moiety is an alkyl substituted with anaryl (e.g., phenylmethyl (benzyl)). The term “alkyl” also includes theside chains of natural and unnatural amino acids.

The term “aryl” includes groups, e.g., 5- and 6-membered single-ringaromatic groups, that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles,” “heteroaryls” or “heteroaromatics.” The aromaticring can be substituted at one or more ring positions with suchsubstituents as described above, as for example, alkyl, alkenyl,alkynyl, halogen, hydroxyl, alkoxy, aryl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH,alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl,alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can alsobe fused or bridged with alicyclic or heterocyclic rings which are notaromatic so as to form a polycycle (e.g., tetralin). The term heteroarylincludes unsaturated cyclic compounds such as azirine, oxirene,dithiete, pyrroline, pyrrole, furan, dihydrofuran, dihydrothiophene,thiophene, pyrazole, imidazole, oxazole, thiazole, isothiazole,12,2,3-triazole, 1,2,4, triazole, dithiazole, tetrazole, pyridine,pyran, pyrimidine, pyran, thiapyrane, diazine, thiazine, dioxine,triazine and tetrazene.

The term “heterocyclic moiety” includes saturated cyclic moieties havinga closed ring of atoms in which at least one atom is not a carbon. Asused herein, heterocyclic moieties do not include heteroaryl moieties,in which the closed ring of atoms is both heterocyclic and aromaticand/or unsaturated. Examples of heterocyclic moieties include aziridine,ethylene oxide, thiirane, dioxirane, azetidine, oxetane, thietane,dioxetane, dithietane, pyrrolidine, tetrahydrofuran,tetrahydrothiophene, imidazolidine, oxazolidine, thiazolidine,dioxolane, dithiolane, piperidine, tetrahydropyran, thiane, piperzine,pxazine, dithiane, dioxane and trioxane.

The term “heterocyclic moiety” includes both “unsubstituted heterocyclicmoieties” and “substituted heterocyclic moieties,” the latter of whichincludes moieties having substituents replacing a hydrogen on one ormore of the atoms on the closed ring. Such substituents can include, forexample, alkyl, alkenyl, alkynyl, halogens, hydroxyl, arylalkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyl oxy,aryloxycarbonyloxy, —COOH, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond. For example, the term “alkenyl”includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl,butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.),branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups(cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl,cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenylgroups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. Theterm “alkenyl” further includes alkenyl groups which include oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone. In certain embodiments, a straight chain orbranched chain alkenyl group has 6 or fewer carbon atoms in its backbone(e.g., C₂-C₆ or straight chain, C₃-C₆ for branched chain). Likewise,cycloalkenyl groups may have from 3-8 carbon atoms in their ringstructure. The term C₂-C₆ includes alkenyl groups containing 2 to 6carbon atoms.

Moreover, the term “alkenyl” includes both “unsubstituted alkenyls” and“substituted alkenyls,” the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogens, hydroxyl, aryl alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyl oxy, aryloxycarbonyloxy, —COOH,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond. For example, the term “alkynyl”includes straight-chain alkynyl groups (e.g., ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.),branched-chain alkynyl groups, and cycloalkyl or cycloalkenylsubstituted alkynyl groups. The term “alkynyl” further includes alkynylgroups which include oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more carbons of the hydrocarbon backbone. In certainembodiments, a straight chain or branched chain alkynyl group has 6 orfewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). The term C₂-C₆ includes alkynyl groupscontaining 2 to 6 carbon atoms.

Moreover, the term “alkynyl” includes both “unsubstituted alkynyls” and“substituted alkynyls,” the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogens, hydroxyl, aryl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—). It also includes substituted acyl moieties. The term“substituted acyl” includes acyl groups where one or more of thehydrogen atoms are replaced by for example, alkyl, alkenyl, alkynyl,halogens, hydroxyl, aryl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “acylamino” includes moieties wherein: an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The terms “alkoxyalkyl,” “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups.

Examples of substituted alkoxy groups include halogenated alkoxy groups.The alkoxy groups can be substituted with groups such as alkyl, alkenyl,alkynyl, halogen, hydroxyl, aryl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The termincludes “alkyl amino” which comprises groups and compounds wherein: thenitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein: the nitrogen atom is bound toat least two additional alkyl groups. The term “arylamino” and“diarylamino” include groups in which the nitrogen is bound to at leastone or two aryl groups, respectively. The term “alkylarylamino,”“alkylaminoaryl” or “arylaminoalkyl” refers to an amino group which isbound to at least one alkyl group and at least one aryl group. The term“alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl group bound to anitrogen atom which is also bound to an alkyl group.

The term “amide,” “amido” or “aminocarbonyl”” includes compounds ormoieties which contain a nitrogen atom which is bound to the carbon of acarbonyl or a thiocarbonyl group. The term includes “alkaminocarbonyl”or “alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl oralkynyl groups bound to an amino group bound to a carbonyl group. Itincludes arylaminocarbonyl and arylcarbonylamino groups, which includearyl or heteroaryl moieties bound to an amino group that is bound to thecarbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarbonyl,” “alkenylaminocarbonyl,” “alkynylaminocarbonyl,”“arylaminocarbonyl,” “alkylcarbonylamino,” “alkenyl carbonylamino,”“alkynylcarbonylamino,” and “arylcarbonylamino” are included in term“amide.” Amides also include urea groups (aminocarbonylamino) andcarbamates (oxycarbonylamino).

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom. Thecarbonyl can be further substituted with any moiety which allows thecompounds of the invention to perform its intended function. Forexample, carbonyl moieties may be substituted with alkyls, alkenyls,alkynyls, aryls, alkoxy, aminos, etc. Examples of moieties which containa carbonyl include aldehydes, ketones, carboxylic acids, amides, esters,anhydrides, etc. The term “carboxy” further includes the structure of—COOH and —COO⁻.

The term “oximyl” includes compounds and moieties that contain a carbonconnected with a double bond to a nitrogen atom, which is, in turnconnected to a hydroxyl or an alkoxyl group. The term “hydrazinyl”includes compounds and moieties that contain a carbon connected with adouble bond to a nitrogen atom, which is, in turn, connected to an aminogroup.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to, alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and “alkthioalkynyl” refer to compounds or moieties inwhich an alkyl, alkenyl or alkynyl group is bonded to a sulfur atom thatis covalently bonded to an alkenyl or alkynyl group, respectively.

The term “sulfonyl” includes moieties containing a sulfonyl functionalgroup (e.g., SO₂) attached to two carbons via a covalent bond to thesulfur atom of the sulfonyl functional group.

The term “hydroxyl” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

II. Screening Assays

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptidomimetics, small molecules or other drugs) whichmodulate, for example one or more biological activities of anRNA-binding protein, e.g., the ability to 1) interact, e.g., bind, e.g.,form a complex, with an RNA molecule, e.g., an RNA molecule comprisingan RNA-binding recognition element of the RNA-binding protein, 2)modulate embryogenesis, 3) modulate cell viability, 4) modulate anteriorpatterning, 5) modulate germ cell totipotency, 6) modulate developmentof the intestine, 7) modulate development of germline blastomeres, 8)modulate development of pharyngeal tissue, 9) modulate expression and/oractivity of a gene known to be directly or indirectly regulated by theRNA-binding protein, e.g., PAL-1, NOS-2, APX-1 protein, and GLD-1, 10)modulate tubercle formation, or for testing or optimizing the activityof such agents.

The assays can be used to identify agents that modulate the function ofan RNA-binding protein or a molecule in a signal transduction pathwayinvolving the RNA-binding protein. The function of an RNA-bindingprotein can be affected at any level, including transcription, proteinexpression, protein localization, and/or cellular activity. The subjectassays can also be used to identify, e.g., agents that alter theinteraction of an RNA-binding protein with a binding partner, e.g., anRNA molecule comprising an RNA-binding protein recognition element, ormodulate, e.g., inhibit, the stability of such interaction.

The subject screening assays can measure the activity of an RNA-bindingprotein directly (e.g., formation of a complex with an RNA moleculecomprising an RNA-binding protein recognition element), or can measure adownstream event controlled by modulation of the RNA-binding protein(e.g., embryogenesis, cell differentiation, expression and/or activityof, a gene known to be directly or indirectly regulated by theRNA-binding protein, e.g., PAL-1, NOS-2, APX-1 protein, and GLD-1).

The subject screening assays employ indicator compositions. Theseindicator compositions comprise the components required for performingan assay that detects and/or measures a particular event. The indicatorcompositions of the invention provide a reference readout and changes inthe readout can be monitored in the presence of one or more testcompounds. A difference in the readout in the presence and the absenceof the compound indicates that the test compound is a modulator of themolecule(s) present in the indicator composition.

The indicator composition used in the screening assay can be a cell thatexpresses an RNA-binding protein and/or an RNA molecule comprising anRNA-binding protein recognition element. For example, a cell thatnaturally expresses or, more preferably, a cell that has been engineeredto express the protein and/or the RNA molecule comprising an RNA-bindingprotein recognition element by introducing into the cell an expressionvector encoding the protein may be used. The cell may be a helminthcell, a plant cell, a yeast cell, a bacterial cell, or a mammalian cell,e.g., a human cell. Alternatively, the indicator composition can be acell-free composition that includes the protein and/or the RNA moleculecomprising an RNA-binding protein recognition element (e.g., a cellextract or a composition that includes e.g., either purified natural orrecombinant protein and/or RNA).

The indicator compositions used in the screening assays of the inventioncan be a cell that expresses an RNA-binding protein or biologicallyactive fragment thereof, e.g., a fragment of the protein that interacts,e.g., binds, to an RNA-binding protein recognition element, e.g., afragment comprising a CCCH-type tandem zinc finger or a KH domain.

In another embodiment, the indicator composition comprises more than onepolypeptide. For example, in one embodiment the subject assays areperformed in the presence of more than one RNA-binding protein, e.g.,MEX-5, GLP-1, NOS-2, MEX-6, POS-1, MEX-3, PAL-1. It will be understoodthat in addition to the recited proteins, e.g., helminth proteins, e.g.,nematode proteins, suitable proteins for use in the methods of theinvention include plant and mammalian homologues of such proteins, e.g.,TTP, the mammalian homologue of MEX-5. One of ordinary skill in the artcan identify such proteins based on sequence and/or database and/orhomology searching and analyses.

Compounds that modulate the expression and/or activity of an RNA-bindingprotein, identified using the assays described herein can be useful fortreating a subject that would benefit from the modulation of expressionand/or activity of the RNA-binding protein, e.g., a subject with aparasitic associate state.

In one embodiment, secondary assays can be used to confirm that themodulating agent affects the RNA-binding protein molecule in a specificmanner. For example, compounds identified in a primary screening assaycan be used in a secondary, tertiary, etc. screening assay to determinewhether the compound affects an RNA-binding protein-related activity asdescribed herein. Accordingly, in another aspect, the invention pertainsto a combination of two or more of the assays described herein. Forexample, a modulating agent can be identified using a cell-based or acell-free assay, e.g., to detect an interaction, e.g., formation of acomplex, and the ability of the agent to modulate the activity of theRNA-binding protein or a molecule involved in a signal transductionpathway involving the RNA-binding protein can be confirmed using abiological read-out to measure, e.g., embryogenesis, an immune response,e.g., cytokine production, in vitro or in vivo.

Moreover, a modulator of an RNA-binding protein expression and/oractivity identified as described herein (e.g., a small molecule) may beused in an animal model and/or plant model to determine the efficacy,toxicity, or side effects of treatment with such a modulator.Alternatively, a modulator identified as described herein may be used inan animal model to determine the mechanism of action of such amodulator. An example of a nematode parasite model that may be used toevaluate the efficacy of treatment of nematode parasitism with an RNAbinding modulatory compound identified herein is described, for example,in Lok J. B. (“Strongyloides stercoralis: a model for translationalresearch on parasitic nematode biology” 2007 WormBook, ed. The C.elegans Research Community, WormBook, doi/10.1895/wormbook.1.134.1, atwww.wormbook.org), the entire contents of which is incorporated hereinby reference. S. stercoralis is a significant pathogen of humans and canbe maintained in laboratory dogs and gerbils. Examples of animal modelsthat may be used to determine the efficacy of treatment of nematodeparasitism with an RNA binding modulatory compound identified herein aredescribed, for example, in Camberis, M. et al., (Animal Model ofNippostrongylus brasiliensis and Heligmosomoides polygyrus, 2003 CurrentProtocols in Immunology 19.12.1-19.12.27), the entire contents of whichis incorporated herein by reference.

In one embodiment, the screening assays of the invention are highthroughput or ultra high throughput (e.g., Fernandes, P. B., Curr OpinChem. Biol. 1998 2:597; Sundberg, S A, Curr Opin Biotechnol. 2000,11:47).

Exemplary cell based and cell free assays of the invention are describedin more detail below.

Cell Based Assays

The indicator compositions of the invention may be cells that express anRNA-binding protein and/or an RNA molecule comprising an RNA-bindingprotein recognition element. For example, a cell that naturallyexpresses endogenous polypeptide and/or RNA molecule, or, morepreferably, a cell that has been engineered to express one or moreexogenous polypeptides and/or RNA molecules, e.g., by introducing intothe cell an expression vector encoding the protein may be used in a cellbased assay.

The cells used in the instant assays can be eukaryotic or prokaryotic inorigin. For example, in one embodiment, the cell is a bacterial cell. Inone embodiment, the cell is a helminth, e.g., nematode, cell. In anotherembodiment, the cell is a plant cell. In another embodiment, the cell isa fungal cell, e.g., a yeast cell. In another embodiment, the cell is avertebrate cell, e.g., an avian or a mammalian cell (e.g., a murinecell, rat cell, rhesus monkey, or a human cell). In another embodiment,the cell is a human cell.

In another embodiment, cells for use in the methods of the invention arederived from a cell line, preferably one which expresses low levels ofendogenous RNA-binding protein and/or an RNA molecule comprising anRNA-binding protein recognition element and is then engineered toexpress recombinant protein and/or RNA.

Recombinant expression vectors that may be used for expression ofpolypeptides are known in the art. For example, the cDNA is firstintroduced into a recombinant expression vector using standard molecularbiology techniques. A cDNA can be obtained, for example, byamplification using the polymerase chain reaction (PCR) or by screeningan appropriate cDNA library.

When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma virus, adenovirus,cytomegalovirus and Simian Virus 40. Non-limiting examples of mammalianexpression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) andpMT2PC (Kaufman, et al. (1987), EMBO J. 6:187-195). A variety ofmammalian expression vectors carrying different regulatory sequences arecommercially available.

Vector DNA may be introduced into cells via conventional transfectiontechniques. As used herein, the various forms of the term “transfection”are intended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into host cells, includingcalcium phosphate co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transfecting hostcells can be found in Sambrook, et al. (Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), andother laboratory manuals.

For stable transfection of cells, it is known that, depending upon theexpression vector and transfection technique used, only a small fractionof cells may integrate the foreign DNA into their genome. In order toidentify and select these integrants, a gene that encodes a selectablemarker (e.g., resistance to antibiotics) is generally introduced intothe host cells along with the gene of interest. Preferred selectablemarkers include those which confer resistance to drugs, such as G418,hygromycin and methotrexate. Nucleic acid encoding a selectable markercan be introduced into a host cell on a separate vector from thatencoding an RNA-binding protein and/or an RNA molecule comprising anRNA-binding protein recognition element, on the same vector. Cellsstably transfected with the introduced nucleic acid can be identified bydrug selection (e.g., cells that have incorporated the selectable markergene will survive, while the other cells die).

In one embodiment, within the expression vector coding sequences areoperatively linked to regulatory sequences that allow for constitutiveexpression of the molecule in the indicator cell (e.g., viral regulatorysequences, such as a cytomegalovirus promoter/enhancer, may be used).Use of a recombinant expression vector that allows for constitutiveexpression of the genes in the indicator cell is preferred foridentification of compounds that enhance or inhibit the activity of themolecule. In an alternative embodiment, within the expression vector thecoding sequences are operatively linked to regulatory sequences of theendogenous gene (i.e., the promoter regulatory region derived from theendogenous gene). Use of a recombinant expression vector in whichexpression is controlled by the endogenous regulatory sequences ispreferred for identification of compounds that enhance or inhibit thetranscriptional expression of the molecule.

For example, an indicator cell can be transfected with an expressionvector comprising an RNA-binding protein, or biologically activefragment thereof, incubated in the presence and in the absence of a testcompound, and the effect of the compound on the expression and/oractivity of the molecule or on a biological response regulated by theRNA-binding protein, e.g., an RNA-binding protein-related activity, canbe determined. The biological activities of an RNA-binding proteininclude activities determined in vivo, or in vitro, according tostandard techniques. Activity can be a direct activity, such as anassociation with a target molecule (e.g., an RNA molecule comprising anRNA-binding protein recognition element). Alternatively, activity may bean indirect activity, such as, for example, a cellular signalingactivity occurring downstream of the interaction of the protein with atarget molecule or a biological effect occurring as a result of thesignaling cascade triggered by that interaction, such as embryogenesisand/or cell differentiation.

Compounds that modulate RNA-binding protein production, expressionand/or activity of may be identified using various “read-outs.”

For example, in one embodiment, gene expression of an RNA-bindingprotein can be measured. In another embodiment, expression of a genecontrolled by an RNA-binding protein can be measured.

In another embodiment, protein expression may be measured. For example,standard techniques such as Western blotting or in situ detection can beused.

In one embodiment a downstream effect of modulation of an RNA-bindingprotein, e.g., the effect of a compound on cell viability and/orembryogenesis, and/or tubercle formation in a plant, may be used as anindicator of modulation of the activity of an RNA-binding protein by,for example, monitoring directly (e.g. by microscopic examination of thecells), or indirectly, e.g., by monitoring one or more markers of, forexample, embryogenesis.

Standard methods for detecting mRNA of interest, such as reversetranscription-polymerase chain reaction (RT-PCR) and Northern blotting,are known in the art. Standard methods for detecting protein secretionin culture supernatants, such as enzyme linked immunosorbent assays(ELISA), are also known in the art. Proteins can also be detected usingantibodies, e.g., in an immunoprecipitation reaction or for staining andFACS analysis.

The ability of the test compound to modulate an RNA-binding moleculeinteraction with a target molecule can also be determined. For example,in one embodiment, the interaction of an RNA-binding molecule and an RNAmolecule comprising an RNA-binding protein recognition element can bemeasured as described in, for example, Pagano, et al. (2007) J. Biol.Chem. 282:8883 and Farley, et al. (2008) RNA 14: 2685. In certainembodiments of the invention, the RNA-binding protein recognitionelement comprises the consensus sequence UA(U₂₋₃)RD(N₁₋₃)G. In stillother embodiments of the invention, the RNA-binding protein recognitionelement comprises the consensus sequence DKAG(N₀₋₃)UHUA. In oneembodiment, the RNA-binding protein recognition element, i.e.,DKAG(N₀₋₃)UHUA, binds with MEX-3. In another embodiment, the RNA-bindingprotein recognition element, i.e., UA(U₂₋₃)RD(N₁₋₃)G, binds with POS-1.

Determining the ability of the test compound to modulate, for example,an RNA-binding molecule, binding to a target molecule (e.g., a targetRNA molecule, e.g., an RNA-binding protein recognition element) can alsobe accomplished, for example, by determining the ability of themolecules to be coimmunoprecipitated or by coupling the target moleculewith a radioisotope or enzymatic label such that binding of the targetmolecule to an RNA-binding molecule or an RNA-bindingmolecule-interacting polypeptide can be determined, e.g., by detectingthe labeled target molecule in a complex. Alternatively, for example, anRNA-binding molecule can be coupled with a radioisotope or enzymaticlabel to monitor the ability of a test compound to modulate anRNA-binding molecule binding to a target molecule in a complex.

Determining the ability of the test compound to interact with anRNA-binding molecule can be accomplished, for example, by coupling thecompound with a radioisotope or enzymatic label such that interaction ofthe compound can be determined by detecting the labeled compound in acomplex. For example, targets can be labeled with 125I, 35S, 14C, or 3H,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,compounds can be labeled, e.g., with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

In another embodiment, fluorescence technologies can be used, e.g.,fluorescence polarization, time-resolved fluorescence, and fluorescenceresonance energy transfer (Selvin, P R, Nat. Struct. Biol. 2000 7:730;Hertzberg R P and Pope A J, Curr Opin Chem Biol. 2000 4:445).

It is also within the scope of this invention to determine the abilityof a compound to interact with an RNA-binding protein without thelabeling of any of the interactants. For example, a microphysiometer maybe used to detect the interaction of a compound with a an RNA-bindingprotein without the labeling of either the compound or the molecule(McConnell, H. M., et al. (1992) Science 257:1906-1912). As used herein,a “microphysiometer” (e.g., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate may be used as an indicator of the interactionbetween compounds.

In yet another aspect of the invention, an RNA-binding protein orfragments thereof may be used as “bait protein” e.g., in a two-hybridassay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos,et al. (1993) Cell 72:223-232; Madura, et al. (11993) J. Biol. Chem.268:12046-12054; Bartel, et al. (1993) Biotechniques 14:920-924;Iwabuchi, et al. (1993) Oncogene 8: 1693-1696; and Brent WO94/10300), toidentify other proteins, which bind to or interact with an RNA-bindingprotein (“binding proteins” or “bp”) and are involved in an RNA-bindingprotein activity. Such proteins are also likely to be involved in thepropagation of signals by the RNA-binding protein. The two-hybrid systemis based on the modular nature of most transcription factors, whichconsist of separable DNA-binding and activation domains. Briefly, theassay utilizes two different DNA constructs. In one construct, the genethat codes for an RNA-binding protein is fused to a gene encoding theDNA binding domain of a known transcription factor (e.g., GAL-4). In theother construct, a DNA sequence, from a library of DNA sequences, thatencodes an unidentified protein (“prey” or “sample”) is fused to a genethat codes for the activation domain of the known transcription factor.If the “bait” and the “prey” proteins are able to interact, in vivo,forming an RNA-binding protein-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g.,LacZ) which is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the protein which interacts with the RNA-binding protein.

Cell-Free Assays

Alternatively, the indicator composition can be a cell-free compositionthat includes an RNA-binding protein, e.g., a cell extract from a cellexpressing the protein or a composition that includes purified eithernatural or recombinant protein.

In one embodiment, the indicator composition is a cell free composition.Polypeptides expressed by recombinant methods in a host cells or culturemedium can be isolated from the host cells, or cell culture medium usingstandard methods for protein purification. For example, ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodies may beused to produce a purified or semi-purified protein that may be used ina cell free composition. Alternatively, a lysate or an extract of cellsexpressing the protein of interest can be prepared for use as cell-freecomposition. Cell extracts with the appropriate post-translationmodifications of proteins can be prepared using commercially availableresources found at, for example Promega, Inc.

In one embodiment, compounds that specifically modulate an activity ofan RNA-binding protein may be identified. For example, compounds thatmodulate an activity of an RNA-binding protein are identified based ontheir ability to modulate the interaction of an RNA-binding protein witha target molecule to which the RNA-binding protein binds, e.g., RNAmolecule comprising an RNA-binding protein recognition element. Suitableassays are known in the art that allow for the detection ofprotein-protein interactions (e.g., immunoprecipitations and the like)or that allow for the detection of interactions between a DNA or RNAbinding protein and a target DNA or RNA sequence (e.g., electrophoreticmobility shift assays, DNAse I footprinting assays and the like). Byperforming such assays in the presence and absence of test compounds,these assays may be used to identify compounds that modulate (e.g.,inhibit or enhance) the interaction of an RNA-binding protein with atarget molecule.

In the methods of the invention for identifying test compounds thatmodulate an interaction between an RNA-binding protein and a targetmolecule, the complete RNA-binding protein may be used in the method,or, alternatively, only portions of the protein may be used. Forexample, an isolated CCH-type tandem zinger finder domain or a KH domainmay be used. An assay may be used to identify test compounds that eitherstimulate or inhibit the interaction between the an RNA-binding proteinand a target molecule. A test compound that stimulates the interactionbetween the protein and a target molecule is identified based upon itsability to increase the degree of interaction as compared to the degreeof interaction in the absence of the test compound and such a compoundwould be expected to, e.g., increase, the activity of an RNA-bindingprotein in the cell. A test compound that inhibits the interactionbetween the protein and a target molecule is identified based upon itsability to decrease the degree of interaction between the protein and atarget molecule as compared to the degree of interaction in the absenceof the compound and such a compound would be expected to, e.g.,decrease, RNA-binding protein activity.

In one embodiment, the amount of binding of an RNA-binding protein to anRNA molecule comprising an RNA-binding protein recognition element inthe presence of the test compound is greater than the amount of bindingin the absence of the test compound, in which case the test compound isidentified as a compound that enhances binding of an RNA-binding proteinto an RNA molecule comprising an RNA-binding protein recognitionelement. In another embodiment, the amount of binding of the RNA-bindingprotein to an RNA molecule comprising an RNA-binding protein recognitionelement in the presence of the test compound is less than the amount ofbinding of an RNA-binding protein to an RNA molecule comprising anRNA-binding protein recognition element in the absence of the testcompound, in which case the test compound is identified as a compoundthat inhibits binding of an RNA-binding protein to an RNA moleculecomprising an RNA-binding protein recognition element.

For example, interaction, e.g., formation of a complex, of the testcompound to an RNA-binding protein can be determined either directly orindirectly as described above. Determining the ability of an RNA-bindingprotein to interact with a test compound can also be accomplished usinga technology such as real-time Biomolecular Interaction Analysis (BIA)(Sjolander, S, and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345;Szabo, et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). As usedherein, “BIA” is a technology for studying biospecific interactions inreal time, without labeling any of the interactants (e.g., BIAcore).Changes in the optical phenomenon of surface plasmon resonance (SPR) maybe used as an indication of real-time reactions between biologicalmolecules.

In one embodiment of the above assay methods, it may be desirable toimmobilize either an RNA-binding protein or an RNA molecule comprisingan RNA-binding protein recognition element for example, to facilitateseparation of complexed from uncomplexed forms of one or both of themolecules, or to accommodate automation of the assay. Binding to asurface can be accomplished in any vessel suitable for containing thereactants. Examples of Such vessels include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided in which a domain that allows one or both of theproteins to be bound to a matrix is added to one or more of themolecules. For example, glutathione-S-transferase fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or RNA-binding protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix is immobilized in the case of beads, and complex formation isdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of binding or activity determined using standardtechniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, proteins may beimmobilized utilizing conjugation of biotin and streptavidin.Biotinylated protein or target molecules can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies which are reactive with protein ortarget molecules but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andunbound target or SLIM protein is trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with anRNA-binding protein.

Test Compounds

A variety of test compounds can be evaluated using the screening assaysdescribed herein. The term “test compound” includes any reagent or testagent which is employed in the assays of the invention and assayed. Morethan one compound, e.g., a plurality of compounds, can be tested at thesame time in a screening assay. The term “screening assay” preferablyrefers to assays which test the ability of a plurality of compounds toinfluence the readout of choice rather than to tests which test theability of one compound to influence a readout. Preferably, the subjectassays identify compounds not previously known to have the effect thatis being screened for. In one embodiment, high throughput screening maybe used to assay for the activity of a compound.

In certain embodiments, the compounds to be tested can be derived fromlibraries (i.e., are members of a library of compounds). While the useof libraries of peptides is well established in the art, new techniqueshave been developed which have allowed the production of mixtures ofother compounds, Such as benzodiazepines (Bunin, et al. (1992). J. Am.Chem. Soc. 114:10987; DeWitt et al. (1993). Proc. Natl. Acad. Sci., USA90:6909) peptoids (Zuckerman. (1994). J. Med. Chem. 37:2678)oligocarbamates (Cho, et al. (1993). Science. 261:11303), and hydantoins(DeWitt, et al. supra). An approach for the synthesis of molecularlibraries of small organic molecules with a diversity of 104-105 as beendescribed (Carell, et al. (1994). Angew. Chem. Int. Ed. Engl. 33:2059;Carell, et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061).

The compounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries, synthetic library methods requiringdeconvolution, the ‘one-bead one-compound’ library method, and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.12:145). Other exemplary methods for the synthesis of molecularlibraries can be found in the art, for example in: Erb, et al. (1994).Proc. Natl. Acad. Sci., USA 91:11422-; Horwell, et al. (1996)Immunopharmacology 33:68-; and in Gallop, et al. (1994); J. Med. Chem.37:1233.

Exemplary compounds which can be screened for activity include, but arenot limited to, peptides, nucleic acids, carbohydrates, small organicmolecules, and natural product extract libraries.

Candidate/test compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam, K. S., et al. (1991) Nature354:82-84; Houghten, R., et al. (1991) Nature 354:84-86) andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids; 2) phosphopeptides (e.g., members of randomand partially degenerate, directed phosphopeptide libraries, see, e.g.,Songyang, Z., et al. (1993) Cell 72:767-778); 3) antibodies (e.g.,antibodies (e.g., intracellular, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric, and single chain antibodies as well as Fab,F(ab′)2, Fab expression library fragments, and epitope-binding fragmentsof antibodies); 4) small organic and inorganic molecules (e.g.,molecules obtained from combinatorial and natural product libraries); 5)enzymes (e.g., endoribonucleases, hydrolases, nucleases, proteases,synthatases, isomerases, polymerases, kinases, phosphatases,oxido-reductases and ATPases), and 6) mutant forms of molecules.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or Solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al. (1993) Proc. Natl.Acad. Sci., U.S.A. 90:6909; Erb, et al. (1994) Proc. Natl. Acad. Sci.,USA 91:11422; Zuckermann, et al. (1994) J. Med. Chem. 37:2678; Cho, etal. (1993) Science 261:1303; Carrell, et al. (1994) Angew. Chem. Int.Ed. Engl. 33:2059; Carell, et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and Gallop, et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds can be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull, et al. (1992) Proc. Natl. Acad. Sci., USA 89:1865-1869) or phage(Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla, et al. (1990) Proc. Natl. Acad. Sci., USA87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

Compounds identified in the subject screening assays may be used, e.g.,in methods of modulating embryogenesis, a parasitic associated state. Itwill be understood that it may be desirable to formulate suchcompound(s) as pharmaceutical compositions (described supra) prior tocontacting them with cells.

Once a test compound is identified that directly or indirectlymodulates, e.g., inhibits, an RNA-binding protein activity by one of thevariety of methods described herein, the selected test compound (or“compound of interest”) can then be further evaluated for its effect oncells, for example by contacting the compound of interest with cellseither in vivo (e.g., by administering the compound of interest to asubject) or ex vivo (e.g., by isolating cells from the subject andcontacting the isolated cells with the compound of interest or,alternatively, by contacting the compound of interest with a cell line)and determining the effect of the compound of interest on the cells, ascompared to an appropriate control (such as untreated cells or cellstreated with a control compound, or carrier, that does not modulate thebiological response).

The instant invention also pertains to compounds identified in thesubject screening assays.

III. Pharmaceutical Compositions

The invention also pertains to pharmaceutical compositions comprising atherapeutically effective amount of an RNA binding modulatory compound,e.g., a compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V,VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound ofTable 1 or 2, and a pharmaceutically acceptable carrier. Each of thesecompounds may be used alone or in combination as a part of apharmaceutical composition of the invention.

In one embodiment, the RNA binding modulatory compound, e.g., compoundof formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, isadministered in combination with an additional agent, e.g., ananti-helminthic agent. The language “in combination with” an additionalagent, e.g., an antihelminthic agent, includes co-administration of thecompound with an additional agent, e.g., an antihelminthic agent,administration of the compound first, followed by administration of anadditional agent, e.g., an antihelminthic agent, and administration ofan additional agent, e.g., antihelminthic agent first, followed byadministration of the compound. The compound can be administeredsubstantially at the same time as the additional agent, e.g.,antihelminthic agent, or at substantially different times as theadditional agent, e.g., antihelminthic agent. Optimal administrationrates for a given protocol of administration of the compound and/or theadditional agent, e.g., antihelminthic agent, can be readily ascertainedby those skilled in the art using conventional dosage determinationtests conducted with regard to the specific compounds being utilized,the particular compositions formulated, the mode of application, theparticular site of administration and the like.

The phrases “anti-helmintic agent” and “anti-helminthic agent,” usedinterchangeably herein, include compounds that expel parasitic wormsfrom the body of a subject. In one embodiment, the anti-helmintic agentis a vermifuge agent (e.g., a compound that stuns the parasitic wormprior to or substantially at the same time as the expelling). In anotherembodiment, the anti-helmintic agent is a vermicide (e.g., a compoundthat kills the parasitic worm prior to or substantially at the same timeas the expelling). Examples of anti-helmintic agents include, but arenot limited to, abamectin (e.g., Affirm®, Agri-Mek®, Avermectin®, Avid®,Vertimec® or Zephyr®), praziquantel (e.g., Biltricide®), albendazole(e.g., Albenza®, Eskazole® or Zentel®), diethylcarbamazine (e.g.,Hetrazan®, Carbilazine®, Caricide®, Cypip®, Ethodryl®, Notezine®,Spatonin®, Filaribits® or Banocide Forte®), mebendazole (e.g., Ovex®,Vermox® or Antiox®), niclosamide (e.g., Niclocide®), ivermectin (e.g.,Stromectol®, Mectizan® or Ivexterm®), suramin (e.g., Germanin®),thiabendazole (e.g., Mintezol®, Tresaderm® or Arbotect®), pyrantelpamoate, levamisole (e.g., Ergamisol®), triclabendazole, (e.g.,Fasonex®) flubendazole (e.g., Flutelmium®, Flubenol®, Biovermin® orFlumoxal®), fenbendazole (e.g., Panacur®, Safe-Guard® or PanacurRabbit®), octadepsipeptide (e.g., emodepside), piperazine derivatives,amino acetonitrile derivatives (e.g., monepantel, Zolvix®), oxamniquine(e.g., Vansil® or Mansil®), metrifonate (e.g., trichlorfon), bithionol,niridazole (e.g., Ambilgar®), stibophen, ciclobendazole, oxantel,pyrvinium, bephenium, desapidin and dichlorophen. One of skill in theart using conventional medical diagnoses would be able to determine theappropriate anti-helmintic agent to administer in combination with theRNA binding modulatory compound, e.g., the compounds of a compound offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2.

The RNA binding modulatory compounds, e.g., compounds of formula I, Ia,Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc,VIId, VIIe, VIIf or a compound of Table 1 or 2, that are basic in natureare capable of forming a wide variety of pharmaceutically acceptablesalts with various inorganic and organic acids. The acids that may beused to prepare pharmaceutically acceptable acid addition salts of thecompounds disclosed herein that are basic in nature are those that formnon-toxic acid addition salts, i.e., salts containing pharmaceuticallyacceptable anions, such as the hydrochloride, hydrobromide, hydroiodide,nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate,acetate, lactate, salicylate, citrate, acid citrate, tartrate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucaronate, saccharate, formate, benzoate,glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate and palmoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts. Although such saltsmust be pharmaceutically acceptable for administration to a subject,e.g., a mammal, it is often desirable in practice to initially isolatethe compound from the reaction mixture as a pharmaceuticallyunacceptable salt and then simply convert the latter back to the freebase compound by treatment with an alkaline reagent and subsequentlyconvert the latter free base to a pharmaceutically acceptable acidaddition salt. The acid addition salts of the base compounds are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained. The preparation of other compounds not specificallydescribed in the experimental section can be accomplished usingcombinations of the described reactions that will be apparent to thoseskilled in the art.

The RNA binding modulatory compounds, e.g., compounds of formula I, Ia,Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc,VIId, VIIe, VIIf or a compound of Table 1 or 2, that are acidic innature are capable of forming a wide variety of pharmaceuticallyacceptable base salts. The chemical bases that may be used as reagentsto prepare pharmaceutically acceptable base salts of those compoundsthat are acidic in nature are those that form non-toxic base salts withsuch compounds. Such non-toxic base salts include, but are not limitedto those derived from such pharmaceutically acceptable cations such asalkali metal cations (e.g., potassium and sodium) and alkaline earthmetal cations (e.g., calcium and magnesium), ammonium or water-solubleamine addition salts such as N-methylglucamine-(meglumine), and thelower alkanolamnionium and other base salts of pharmaceuticallyacceptable organic amines. The pharmaceutically acceptable base additionsalts of the compounds that are acidic in nature may be formed withpharmaceutically acceptable cations by conventional methods. Thus, thesesalts may be readily prepared by treating the compounds disclosed hereinwith an aqueous solution of the desired pharmaceutically acceptablecation and evaporating the resulting solution to dryness, preferablyunder reduced pressure. Alternatively, a lower alkyl alcohol solution ofthe compounds of the invention may be mixed with an alkoxide of thedesired metal and the solution subsequently evaporated to dryness.

The term “pharmaceutically acceptable carrier” includes any carrier thatis suitable for administration to a mammal.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microbes may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin. In some cases, in order to prolong the effect of a drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

Pharmaceutical compositions of the present invention may be administeredto a subject, e.g., a non-human animal or a human, orally, parenterally,topically, rectally, nasally, intravaginally or intracisternally. Theyare, of course, given by forms suitable for each administration route.For example, they are administered in tablets or capsule form, byinjection, inhalation, eye lotion, ointment, etc., administration byinjection, infusion or inhalation; topical by lotion or ointment; andrectal or vaginal suppositories.

The phrases “parenteral administration” and “administered parenterally”as used herein include modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally,” as usedherein, includes the administration of the RNA binding modulatorycompound, e.g., compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III,IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIe, VIId, VIIe, VIIf or acompound of Table 1 or 2, other than directly into the central nervoussystem, such that it enters the subject's system and, thus, is subjectto metabolism and other like processes, for example, subcutaneousadministration.

In some methods, the compositions of the invention can be topicallyadministered to any epithelial surface. An “epithelial surface” includesan area of tissue that covers external surfaces of a body, or whichlines hollow structures including, but not limited to, cutaneous andmucosal surfaces. Such epithelial surfaces include oral, pharyngeal,esophageal, pulmonary, ocular, aural, nasal, buccal, lingual, vaginal,cervical, genitourinary, alimentary, and anorectal surfaces.

Compositions can be formulated in a variety of conventional formsemployed for topical administration. These include, for example,semi-solid and liquid dosage forms, such as liquid solutions orsuspensions, suppositories, douches, enemas, gels, creams, emulsions,lotions, slurries, powders, sprays, lipsticks, foams, pastes,toothpastes, ointments, salves, balms, douches, drops, troches, chewinggums, lozenges, mouthwashes, rinses.

Conventionally used carriers for topical applications include pectin,gelatin and derivatives thereof, polylactic acid or polyglycolic acidpolymers or copolymers thereof, cellulose derivatives such as methylcellulose, carboxymethyl cellulose, or oxidized cellulose, guar gum,acacia gum, karaya gum, tragacanth gum, bentonite, agar, carbomer,bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum,hectorite, ispaghula husk, polyvinypyrrolidone, silica and derivativesthereof, xanthan gum, kaolin, talc, starch and derivatives thereof,paraffin, water, vegetable and animal oils, polyethylene, polyethyleneoxide, polyethylene glycol, polypropylene glycol, glycerol, ethanol,propanol, propylene glycol (glycols, alcohols), fixed oils, sodium,potassium, aluminum, magnesium or calcium salts (such as chloride,carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptateor tartrate).

Standard composition strategies for topical agents can be applied to theRNA binding modulatory compounds of the invention or a pharmaceuticallyacceptable salt thereof in order to enhance the persistence andresidence time of the drug, and to improve the prophylactic efficacyachieved.

For topical application to be used in the lower intestinal tract orvaginally, a rectal suppository, a suitable enema, a gel, an ointment, asolution, a suspension or an insert can be used. Topical transdermalpatches may also be used. Transdermal patches have the added advantageof providing controlled delivery of the compositions of the invention tothe body. Such dosage forms can be made by dissolving or dispersing theagent in the proper medium.

Compositions of the invention can be administered in the form ofsuppositories for rectal or vaginal administration. These can beprepared by mixing the agent with a suitable non-irritating carrierwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum or vagina to release the drug. Suchmaterials include cocoa butter, beeswax, polyethylene glycols, asuppository wax or a salicylate that is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent. Compositions which aresuitable for vaginal administration also include pessaries, tampons,creams, gels, pastes, foams, films, or spray compositions containingsuch carriers as are known in the art to be appropriate. The carrieremployed in the pharmaceutical compositions of the invention should becompatible with vaginal administration.

For ophthalmic applications, the pharmaceutical compositions can beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the compositions can beformulated in an ointment such as petrolatum. Exemplary ophthalmiccompositions include eye ointments, powders, solutions and the like.

Powders and sprays can contain, in addition to the compound of theinvention, carriers such as lactose, talc, aluminum hydroxide, calciumsilicates and polyamide powder, or mixtures of these substances. Sprayscan additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of an RNA binding modulatory compound, e.g.,compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa,VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or2, together with conventional pharmaceutically acceptable carriers andstabilizers. The carriers and stabilizers vary with the requirements ofthe particular compound, but typically include nonionic surfactants(e.g., Tweens, Pluronics, polyethylene glycol and the like), proteinslike serum albumin, sorbitan esters, oleic acid, lecithin, amino acidssuch as glycine, buffers, salts, sugars or sugar alcohols. Aerosolsgenerally are prepared from isotonic solutions. Generation of theaerosol or any other means of delivery of the present invention may beaccomplished by any of the methods known in the art. For example, in thecase of aerosol delivery, the compound is supplied in a finely dividedform along with any suitable carrier with a propellant.

Liquefied propellants are typically gases at ambient conditions and arecondensed under pressure. The propellant may be any acceptable and knownin the art including propane and butane, or other lower alkanes, such asthose of up to 5 carbons. The composition is held within a containerwith an appropriate propellant and valve, and maintained at elevatedpressure until released by action of the valve.

Compositions of the invention can also be orally administered in anyorally-acceptable dosage form including, but not limited to, capsules,cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), powders, granules, or as a solutionor a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of sucrose octasulfate and/or antibiotic orcontraceptive agent(s) as an active ingredient. A compound describedherein may also be administered as a bolus, electuary or paste. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added. Tablets, and other soliddosage forms, such as dragees, capsules, pills and granules, may bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.They may also be formulated so as to provide slow or controlled releaseof the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile,other polymer matrices, liposomes and/or microspheres. They may besterilized by, for example, filtration through a bacteria-retainingfilter, or by incorporating sterilizing agents in the form of sterilesolid compositions which can be dissolved in sterile water, or someother sterile injectable medium immediately before use. Thesecompositions may also optionally contain opacifying agents and may be ofa composition that they release the RNA binding modulatory compound,e.g., compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI,VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table1 or 2, only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to compounds of the invention, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Sterile injectable forms of the compositions of this invention can beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a nontoxic parenterally-acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. Fatty acids, such as oleicacid and its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically-acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant. The RNA binding modulatory compound, e.g.,compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa,VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or2, or a pharmaceutically acceptable salt thereof will represent somepercentage of the total dose in other dosage forms in a material forminga combination product, including liquid solutions or suspensions,suppositories, douches, enemas, gels, creams, emulsions, lotionsslurries, soaps, shampoos, detergents, powders, sprays, lipsticks,foams, pastes, toothpastes, ointments, salves, balms, douches, drops,troches, lozenges, mouthwashes, rinses and others.

In one embodiment, the compounds of the invention may be administeredprophylactically. For prophylactic applications, the pharmaceuticalcomposition of the invention can be applied prior to potentialinfection. The timing of application prior to potential infection can beoptimized to maximize the prophylactic effectiveness of the compound.The timing of application will vary depending on the mode ofadministration, doses, the stability and effectiveness of composition,the frequency of the dosage, e.g., single application or multipledosage. One skilled in the art will be able to determine the mostappropriate time interval required to maximize prophylacticeffectiveness of the compound.

An RNA binding modulatory compound, e.g., compound of formula I, Ia, Ib,II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc,VIId, VIIe, VIIf or a compound of Table 1 or 2, when present in acomposition will generally be present in an amount from about 0.000001%to about 100%, more preferably from about 0.001% to about 50%, and mostpreferably from about 0.01% to about 25% of total weight.

For compositions of the present invention comprising a carrier, thecomposition comprises, for example, from about 1% to about 99%,preferably from about 50% to about 99%, and most preferably from about75% to about 99% by weight of at least one carrier.

Also, the separate components of the compositions of the invention maybe preblended or each component may be added separately to the sameenvironment according to a predetermined dosage for the purpose ofachieving the desired concentration level of the treatment componentsand so long as the components eventually come into intimate admixturewith each other. Further, the present invention may be administered ordelivered on a continuous or intermittent basis.

The RNA binding modulatory compounds, e.g., compounds of formula I, Ia,Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc,VIId, VIIe, VIIf or a compound of Table 1 or 2, may be used in theveterinary sector in any technique routine to one of skill in the art,including, for example, oral administration, parenterally (e.g.,intraruminal, intramuscular, intravenous or subcutaneous injection) orby transdermal methods. The RNA binding modulatory compounds, e.g.,compounds of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa,VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or2 may also be dispersed or dissolved in a pharmaceutically acceptablecarrier for injection or transdermal application. Alternatively, the RNAbinding modulatory compounds, e.g., compounds of formula I, Ia, Ib, II,IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId,VIIe, VIIf or a compound of Table 1 or 2, may be formulated into animplant for subcutaneous administration.

For oral administration to warm-blooded animals, the RNA bindingmodulatory compounds, e.g., compounds of formula I, Ia, Ib, II, IIa,IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe,VIIf or a compound of Table 1 or 2, may be formulated as animal feeds,animal feed premixes, animal feed concentrates, pills, pastes,suspensions, solutions, gels, tablets, boluses and capsules. Inaddition, the an RNA binding modulatory compounds, e.g., compounds offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, may beadministered to the animals in their drinking water.

For oral administration, the dosage form chosen should provide theanimal with about 0.01 mg/kg to 100 mg/kg of animal body weight per dayof the RNA binding modulatory compound, e.g., compounds of formula I,Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb,VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2. For parenteraladministration, the dosage form chosen should provide the animal withabout 0.01 mg/kg to 100 mg/kg of animal body weight per day of the RNAbinding modulatory compound, e.g., compound of formula I, Ia, Ib, II,IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId,VIIe, VIIf or a compound of Table 1 or 2.

The RNA binding modulatory compounds, e.g., compounds of formula, Ia,Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc,VIId, VIIe, VIIf or a compound of Table 1 or 2, may also be appliedtopically to the animals in the form of dips, dusts, collars,medallions, sprays and pour-on formulations. For topical application,dips and sprays usually contain about 0.5 ppm to 5,000 ppm or about 1ppm to 3,000 ppm of the RNA binding modulatory compound, e.g., compoundof formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2. Inaddition, the RNA binding modulatory compounds, e.g., compounds offormula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, may beformulated as ear tags for animals, particularly quadrupeds such ascattle and sheep.

Pesticidal Compositions

The invention also pertains to pesticidal compositions comprising apesticidally effective amount of an RNA binding modulatory compound,e.g., compound of formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI,VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table1 or 2, and an agronomically acceptable carrier. Each of these compoundsmay be used alone or in combination as a part of a pesticidalcomposition of the invention.

In one embodiment, the RNA binding modulatory compound, e.g., compoundof formula I, Ia, Ib, II, IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII,VIIa, VIIb, VIIc, VIId, VIIe, VIIf or a compound of Table 1 or 2, isadministered in combination with an agricultural agent. The language “incombination with” an agricultural agent includes co-administration ofthe compound and with an agricultural agent, administration of thecompound first, followed by administration of an agricultural agent, andadministration of an agricultural agent first, followed byadministration of the compound. The compound can be administeredsubstantially at the same time as the agricultural agent or atsubstantially different times as the agricultural agent. Optimaladministration rates for a given protocol of administration of thecompound and/or the agricultural agent can be readily ascertained bythose skilled in the art using conventional determination testsconducted with regard to the specific compounds being utilized, theparticular compositions formulated, the mode of application, theparticular site of administration and the like.

The term “agricultural agent” includes, for example, insecticides,attractants, sterilizing agents, bactericides, acaricides, nematicides,fungicides, growth-regulating substances, fertilizers or herbicides,such as aldimorph, ampropylfos, ampropylfos-potassium, andoprim,anilazine, azaconazole, azoxystrobin, benalaxyl, benodanil, benomyl,benzamacril, benzamacryl-isobutyl, bialaphos, binapacryl, biphenyl,bitertanol, blasticidin-S, bromuconazole, bupirimate, buthiobate,calcium polysulphide, capsimycin, captafol, captan, carbendazim,carboxin, carvon, quinomethionate, chlobenthiazone, chlorfenazole,chloroneb, chloropicrin, chlorothalonil, chlozolinate, clozylacon,cufraneb, cymoxanil, cyproconazole, cyprodinil, cyprofuram, debacarb,dichlorophen, diclobutrazole, diclofluanid, diclomezine, dicloran,diethofencarb, difenoconazole, dimethirimol, dimethomorph, diniconazole,diniconazole-M, dinocap, diphenylamine, dipyrithione, ditalimfos,dithianon, dodemorph, dodine, drazoxolon, edifenphos, epoxiconazole,etaconazole, ethirimol, etridiazole, famoxadon, fenapanil, fenarimol,fenbuconazole, fenfuram, fenitropan, fenpiclonil, fenpropidin,fenpropimorph, fentin acetate, fentin hydroxide, ferbam, ferimzone,fluazinam, flumetover, fluoromide, fluquinconazole, flurprimidol,flusilazole, flusulfamide, flutolanil, flutriafol, folpet,fosetyl-aluminium, fosetyl-sodium, fthalide, fuberidazole, furalaxyl,furametpyr, furcarbonil, furconazole, furconazole-cis, furmecyclox,guazatine, hexachlorobenzene, hexaconazole, hymexazole, imazalil,imibenconazole, iminoctadine, iminoctadine albesilate, iminoctadinetriacetate, iodocarb, ipconazole, iprobenfos (IBP), iprodione,irumamycin, isoprothiolane, isovaledione, kasugamycin, kresoxim-methyl,copper preparations, such as: copper hydroxide, copper naphthenate,copper oxychloride, copper sulphate, copper oxide, oxine-copper andBordeaux mixture, mancopper, mancozeb, maneb, meferimzone, mepanipyrim,mepronil, metalaxyl, metconazole, methasulfocarb, methfuroxam, metiram,metomeclam, metsulfovax, mildiomycin, myclobutanil, myclozolin, nickeldimethyldithiocarbamate, nitrothal-isopropyl, nuarimol, ofurace,oxadixyl, oxamocarb, oxolinic acid, oxycarboxim, oxyfenthiin,paclobutrazole, pefurazoate, penconazole, pencycuron, phosdiphen,pimaricin, piperalin, polyoxin, polyoxorim, probenazole, prochloraz,procymidone, propamocarb, propanosine-sodium, propiconazole, propineb,pyrazophos, pyrifenox, pyrimethanil, pyroquilon, pyroxyfur,quinconazole, quintozene (PCNB), sulphur and sulphur preparations,tebuconazole, tecloftalam, tecnazene, tetcyclasis, tetraconazole,thiabendazole, thicyofen, thifluzamide, thiophanate-methyl, thiram,tioxymid, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol,triazbutil, triazoxide, triichlamide, tricyclazole, tridemorph,triflumizole, triforine, triticonazole, uniconazole, validamycin A,vinclozolin, viniconazole, zarilamide, zineb, ziram, Dagger G, OK-8705,OK-8801,α.-(1,1-dimethylethyl)-β-(2-phenoxyethyl)-1H-1,2,4-triazole-1-ethanol,α-(2,4-dichlorophenyl)-β-fluoro-β-propyl-1H-1,2,4-triazole-1-ethanol,α-(2,4-dichlorophenyl)-β-methoxy-α-methyl-1H-1,2,4-triazole-1-ethanol,α-(5-methyl-1,3-dioxan-5-yl)-β-[[4-(trifluoromethyl)-phenyl]-methylene]-1H-1,2,4-triazole-1-ethanol,(5RS,6RS)-6-hydroxy-2,2,7,7-tetramethyl-5-(1H-1,2,4-triazol-1-yl)-3-octanone,(E)-α-(methoxyimino)-N-methyl-2-phenoxy-phenylacetamide,1-isopropyl{2-methyl-1-[[[1-(4-methylphenyl)-ethyl]-amino]-carbonyl]-propyl}-carbamate,1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-ethanone-O-(phenylmethyl)-oxime,1-(2-methyl-1-naphthalenyl)-1H-pyrrol-2,5-dione,1-(3,5-dichlorophenyl)-3-(2-propenyl)-2,5-pyrrolidindione,1-[(diiodomethyl)-sulphonyl]4-methyl-benzene,1-[[2-(2,4-dichlorophenyl)-1,3-dioxolan-2-yl]-methyl]-1H-imidazole,1-[1-[2-(4-chlorophenyl)-3-phenyloxiranyl]-methyl]-1H-1,2,4-triazole,1-[1-[2-[(2,4-dichlorophenyl)-methoxy]-phenyl]-ethenyl]-1H-imidazole,1-methyl-5-nonyl-2-(phenylmethyl)-3-pyrrolidinole.2′,6′-dibromo-2-methyl-4′-trifluoromethoxy-4′-trifluoro-methyl-1,3-thiazole-5-carboxanilide,2,2-dichloro-N-[1-(4-chlorophenyl)-ethyl]-1-ethyl-3-methyl-cyclopropanecarboxamide,2,6-dichloro-5-(methylthio)-4-pyrimidinyl-thiocyanate,2,6-dichloro-N-(4-trifluoromethylbenzyl)-benzamide,2,6-dichloro-N-[[4-(trifluoromethyl)-phenyl]-methyl]-benzamide,2-(2,3,3-triiodo-2-propenyl)-2H-tetrazole,2-[(1-methylethyl)-sulphonyl]-5-(trichloromethyl)-1,3,4-thiadiazole,2-[[6-deoxy-4-O-(4-O-methyl-β-D-glycopyranosyl)-α-D-glucopyranosyl]-amino]-4-methoxy-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile,2-aminobutane, 2-bromo-2-(bromomethyl)-pentanedinitrile,2-chloro-N-(2,3-dihydro-1,1,3-trimethyl-1H-inden-4-yl)-3-pyridinecarboxamide,2-chloro-N-(2,6-dimethylphenyl)-N-(isothiocyanatomethyl)-acetamide,2-phenylphenol (OPP),3,4-dichloro-1-[4-(difluoromethoxy)-phenyl]-1H-pyrrol-2,5-dione,3,5-dichloro-N-[cyano[(1-methyl-2-propynyl)-oxy]-methyl]-benzamide,3-(1,1-dimethylpropyl-1-oxo-1H-indene-2-carbonitrile,3-[2-(4-chlorophenyl)-5-ethoxy-3-isoxazolidinyl]-pyridine,4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)-1H-imidazole-1-sulphonamide,4-methyl-tetrazolo[1,5-a]quinazolin-5(4H)-one,8-(1,1-dimethylethyl)-N-ethyl-N-propyl-1,4-dioxaspiro[4,5]decane-2-methanamine,8-hydroxyquinoline sulphate,9H-xanthene-2-[(phenylamino)-carbonyl]-9-carboxylic hydrazide,bis-(1-methylethyl)-3-methyl-4-[(3-methylbenzoyl)-oxy]-2,5-thiophenedicarboxylate,cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-cycloheptanol,cis-4-[3-[4-(1,1-dimethylpropyl)-phenyl-2-methylpropyl]-2,6-dimethyl-morpholinehydrochloride, ethyl[(4-chlorophenyl)-azo]-cyanoacetate, potassiumbicarbonate, methanetetrathiol-sodium salt, methyl1-(2,3-dihydro-2,2-dimethyl-1H-inden-1-yl)-1H-imidazole-5-carboxylate,methyl N-(2,6-dimethylphenyl)-N-(5-isoxazolylcarbonyl)-DL-alaninate,methyl N-(chloroacetyl)-N-(2,6-dimethylphenyl)-DL-alaninate,N-(2,3-dichloro-4-hydroxyphenyl)-1-methylcyclohexanecarboxamide,N-(2,6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-furanyl)-acetamide,N-(2,6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-thienyl)-acetamide,N-(2-chloro-4-nitrophenyl)-4-methyl-3-nitro-benzenesulphonamide,N-(4-cyclohexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine,N-(4-hexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine,N-(5-chloro-2-methylphenyl)-2-methoxy-N-(2-oxo-3-oxazoldinyl)-acetamide,N-(6-methoxy)-3-pyridinyl)-cyclopropanecarboxamide,N-[2,2,2-trichloro-1-[(chloroacetyl)-amino]-ethyl]-benzamide,N-[3-chloro-4,5-bis(2-propinyloxy)-phenyl]-N′-methoxy-methanimidamide,N-formyl-N-hydroxy-DL-alanine-sodium salt,O,O-diethyl[2-(dipropylamino)-2-oxoethyl]-ethylphosphoramidothioate,0-methyl S-phenyl phenylpropylphosphoramidothioate, S-methyl1,2,3-benzothiadiazole-7-carbothioate,spiro[2H]-1-benzopyrane-2,1′(3′H)-isobenzofuran]-3′-one, bronopol,dichlorophen, nitrapyrin, nickel dimethyldithiocarbamate, kasugamycin,octhilinone, furancarboxylic acid, oxytetracyclin, probenazole,streptomycin, tecloftalam, copper sulphate and other copperpreparations, abamectin, acephate, acetamiprid, acrinathrin, alanycarb,aldicarb, aldoxycarb, alpha-cypermethrin, alphamethrin, amitraz,avermectin, AZ 60541, azadirachtin, azamethiphos, azinphos A, azinphosM, azocyclotin, Bacillus popilliae, Bacillus sphaericus, Bacillussubtilis, Bacillus thuringiensis, baculoviruses, Beauveria bassiana,Beauveria tenella, bendiocarb, benfuracarb, bensultap, benzoximate,betacyfluthrin, bifenazate, bifenthrin, bioethanomethrin, biopermethrin,BPMC, bromophos A, bufencarb, buprofezin, butathiofos, butocarboxim,butylpyridaben, cadusafos, carbaryl, carbofuran, carbophenothion,carbosulfan, cartap, chloethocarb, chlorethoxyfos, chlorfenapyr,chlorfenvinphos, chlorfluazuron, chlormephos, chlorpyrifos, chlorpyrifosM, chlovaporthrin, cis-resmethrin, cispermethrin, clocythrin,cloethocarb, clofentezine, cyanophos, cycloprene, cycloprothrin,cyfluthrin, cyhalothrin, cyhexatin, cypermethrin, cyromazine,deltamethrin, demeton M, demeton S, demeton-S-methyl, diafenthiuron,diazinon, dichlorvos, diflubenzuron, dimethoate, dimethylvinphos,diofenolan, disulfoton, docusat-sodium, dofenapyn, eflusilanate,emamectin, empenthrin, endosulfan, Entomopfthora spp., esfenvalerate,ethiofencarb, ethion, ethoprophos, etofenprox, etoxazole, etrimfos,fenamiphos, fenazaquin, fenbutatin oxide, fenitrothion, fenothiocarb,fenoxacrim, fenoxycarb, fenpropathrin, fenpyrad, fenpyrithrin,fenpyroximate, fenvalerate, fipronil, fluazinam, fluazuron,flubrocythrinate, flucycloxuron, flucythrinate, flufenoxuron,flutenzine, fluvalinate, fonophos, fosmethilan, fosthiazate, fubfenprox,furathiocarb, granulosis viruses, halofenozide, HCH, heptenophos,hexaflumuron, hexythiazox, hydroprene, imidacloprid, isazofos,isofenphos, isoxathion, ivermectin, nuclear polyhedrosis viruses,lambda-cyhalothrin, lufenuron, malathion, mecarbam, metaldehyde,methamidophos, Metharhizium anisopliae, Metharhizium flavoviride,methidathion, methiocarb, methomyl, methoxyfenozide, metolcarb,metoxadiazone, mevinphos, milbemectin, monocrotophos, naled, nitenpyram,nithiazine, novaluron, omethoate, oxamyl, oxydemethon M, Paecilomycesfumosoroseus, parathion A, parathion M, permethrin, phenthoate, phorate,phosalone, phosmet, phosphamidon, phoxim, pirimicarb, pirimiphos A,pirimiphos M, profenofos, promecarb, propoxur, prothiofos, prothoate,pymetrozine, pyraclofos, pyresmethrin, pyrethrum, pyridaben,pyridathion, pyrimidifen, pyriproxyfen, quinalphos, ribavirin,salithion, sebufos, silafluofen, spinosad, sulfotep, sulprofos,tau-fluvalinate, tebufenozide, tebufenpyrad, tebupirimiphos,teflubenzuron, tefluthrin, temephos, temivinphos, terbufos,tetrachlorvinphos, theta-cypermethrin, thiamethoxam, thiapronil,thiatriphos, thiocyclam hydrogen oxalate, thiodicarb, thiofanox,thuringiensin, tralocythrin, tralomethrin, triarathene, triazamate,triazophos, triazuron, trichlophenidine, trichlorfon, triflumuron,trimethacarb, vamidothion, vaniliprole, Verticillium lecanii, YI 5302,zeta-cypermethrin, zolaprofos,(1R-cis)-[5-(phenylmethyl)-3-furanyl]-methyl-3-[(dihydro-2-oxo-3(2H)-furanylidene)-methyl]-2,2-dimethylcyclopropanecarboxylate,(3-phenoxyphenyl)-methyl-2,2,3,3-tetramethylcyclopropanecarboxylate,1-[(2-chloro-5-thiazolyl)methyl]tetrahydro-3,5-dimethyl-N-nitro-1,3,5-triazine-2(1H)-imine,2-(2-chloro-6-fluorophenyl)-4-[4-(1,1-dimethylethyl)phenyl]-4,5-dihydro-oxazole,2-(acetyloxy)-3-dodecyl-1,4-naphthalenedione,2-chloro-N-[[[4-(1-phenylethoxy)-phenyl]-amino]-carbonyl]-benzamide,2-chloro-N-[[[4-(2,2-dichloro-1,1-difluoroethoxy)-phenyl]-amino]-carbonyl]-benzamide,3-methylphenyl propylcarbamate,4-[4-(4-ethoxyphenyl)-4-methylpentyl]-1-fluoro-2-phenoxy-benzene,4-chloro-2-(1,1-dimethylethyl)-5-[[2-(2,6-dimethyl-4-phenoxyphenoxy)ethyl]thio]-3(2H)-pyridazinone,4-chloro-2-(2-chloro-2-methylpropyl)-5-[(6-iodo-3-pyridinyl)methoxy]-3(2H)-pyridazinone,4-chloro-5-[(6-chloro-3-pyridinyl)methoxy]-2-(3,4-dichlorophenyl)-3(2H)-pyridazinone,Bacillus thuringiensis strain EG-2348,[2-benzoyl-1-(1,1-dimethylethyl)-hydrazinobenzoic acid,2)-dimethyl-3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4,5]dec-3-en-4-ylbutanoate,[3-[(6-chloro-3-pyridinyl)methyl]-2-thiazolidinyldene]-cyanamide,dihydro-2-(nitromethylene)-2H-1,3-thiazine-3(4H)-carboxaldehyde,ethyl[2-[[1,6-dihydro-6-oxo-1-(phenylmethyl)-4-pyridazinyl]oxy]ethyl]-carbamate,N-(3,4,4-trifluoro-1-oxo-3-butenyl)-glycine,N-(4-chlorophenyl)-3-[4-(difluoromethoxy)phenyl]-4,5-dihydro-4-phenyl-1H-pyrazole-1-carboxamide,N-[(2-chloro-5-thiazolyl)methyl]-N′-methyl-N′-nitro-guanidine,N-methyl-N′-(1-methyl-2-propenyl)-1,2-hydrazinedicarbothioamide,N-methyl-N′-2-propenyl-1,2-hydrazinedicarbothioamide andO,O-diethyl[2-(dipropylamino)-2-oxoethyl]-ethylphosphoramidothioate.

Treatment of the plants and soil with the RNA binding modulatorycompounds, e.g., compounds of formula I, Ia, Ib, II, IIa, IIb, IIc, III,IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIf or acompound of Table 1 or 2, may be carried out directly or by allowing thecompounds to act on the surroundings, environment or storage space bythe customary treatment methods, for example by immersion, spraying,evaporation, fogging, scattering, painting on and, in the case ofpropagation material, in particular in the case of seeds, also byapplying one or more coats.

Depending on the plant species or plant cultivars, their location andgrowth conditions (soils, climate, vegetation period, diet), thetreatment according to the invention may also result in superadditive(“synergistic”) effects. Thus, for example, reduced application ratesand/or a widening of the activity spectrum and/or an increase in theactivity of the substances and compositions to be used, better plantgrowth, increased tolerance to high or low temperatures, increasedtolerance to drought or to water or soil salt content, increasedflowering performance, easier harvesting, accelerated maturation, higherharvest yields, better quality and/or a higher nutritional value of theharvested products, better storage stability and/or processability ofthe harvested products that exceed the effects which were actually to beexpected may occur.

The RNA binding modulatory compounds, e.g., compounds of I, Ia, Ib, II,IIa, IIb, IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId,VIIe, VIIf or a compound of Table 1 or 2, may used in unchanged form ortogether with an agronomically acceptable carrier. The term“agronomically acceptable carrier” includes any carrier suitable foradministration to a plant or soil, for example, customary excipients informulation techniques, such as solutions (e.g., directly sprayable ordilutable solutions), emulsions, (e.g., emulsion concentrates anddiluted emulsions), wettable powders, suspensions, soluble powders,powders, dusts, pastes, soluble powders, granules, suspension-emulsionconcentrates, encapsulation into polymeric materials, coatable pastes,natural and synthetic materials impregnated with active compound andmicroencapsulations in polymeric substances. These formulations areproduced in a known manner, for example by mixing the compounds withagronomically acceptable carrier, such as liquid solvents or solidcarriers, optionally with the use of surfactants, including emulsifiers,dispersants, and/foam-formers.

If the agronomically acceptable carrier is water, it may also possibleto employ, for example, organic solvents as auxiliary solvents. Suitableliquid solvents include, for example, aromatics (e.g., xylene, tolueneand alkylnaphthalenes); chlorinated aromatics or chlorinated aliphatichydrocarbons (e.g., chlorobenzenes, chloroethylenes and methylenechloride); aliphatic hydrocarbons (e.g., cyclohexane); paraffins (e.g.,petroleum fractions, mineral and vegetable oils); alcohols (e.g.,butanol or glycol and also their ethers and esters); ketones (e.g.,acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone)and strongly polar solvents (e.g., dimethylformamide and dimethylsulphoxide).

Suitable solid agronomically acceptable carriers include, for example,ammonium salts and ground natural minerals (e.g., kaolins, clays, talc,chalk, quartz, attapulgite, montmorillonite and diatomaceous earth);ground synthetic minerals (e.g., highly disperse silica, alumina andsilicates); crushed and fractionated natural rocks (e.g., calcite,marble, pumice, sepiolite and dolomite); synthetic granules of inorganicand organic meals; granules of organic material (e.g., sawdust, coconutshells, maize cobs and tobacco stalks);

Suitable emulsifiers and foam-formers include, for example, nonionic andanionic emulsifiers (e.g., polyoxyethylene fatty acid esters,polyoxyethylene fatty alcohol ethers, for example, alkylaryl polyglycolethers, alkylsulphonates, alkyl sulphates and arylsulphonates) proteinhydrolysates.

Suitable dispersants include, for example, lignin-sulphite waste liquorsand methylcellulose.

Tackifiers such as carboxymethylcellulose and natural and syntheticpolymers in the form of powders, granules or latices, such as gumarabic, polyvinyl alcohol and polyvinyl acetate, as well as naturalphospholipids, such as cephalins and lecithins, and syntheticphospholipids, can be used in the formulations. Other additives mayinclude, for example, mineral and vegetable oils.

Colorants such as inorganic pigments, for example, iron oxide, titaniumoxide and Prussian Blue, and organic dyestuffs, such as alizarindyestuffs, azo dyestuffs and metal phthalocyanine dyestuffs, and tracenutrients such as salts of iron, manganese, boron, copper, cobalt,molybdenum and zinc may also be included in the agronomically acceptablecarrier.

The pesticidal compositions may be administered to the plant or soil byany techniques known in the art, including, for example, spraying,atomizing, dusting, scattering, coating or pouring. One of skill in theart would be able to determine the appropriate technique foradministration without undue experimentation according the specific pestto be combated, the specific chemical composition and formulation of thecompound being employed, the method of applying thecompound/formulation, and the locus of treatment.

In one embodiment, the pesticidal compositions comprising an RNA bindingmodulatory compound, e.g., compound of formula I, Ia, Ib, II, IIa, IIb,IIc, III, IV, V, VI, VIa, VIb, VII, VIIa, VIIb, VIIc, VIId, VIIe, VIIfor a compound of Table 1 or 2, may be administered by foliarapplication. In another embodiment, the pesticidal composition may alsoreach the plants through the root system via the soil (systemic action)by drenching the locus of the plant with a liquid preparation or byincorporating the substances into the soil in solid form, e.g., in theform of granules (soil application). In rice cultivations, thesegranules may be dispensed over the flooded paddy field. The pesticidalcompositions of the invention may also be applied to tubers or seedgrain, for example, by soaking, spraying or drenching the seed grain ortubers in a liquid pesticidal composition or by coating the tubers orseed grain with a solid pesticidal composition.

The pesticidal compositions disclosed herein generally comprise between0.1 and 95% by weight of active compound, preferably between 0.5 and90%. Favorable application rates are, in general, 1 g to 2 kg of activesubstance (AS) per hectare (ha), for example, 10 g to 1 kg AS/ha or 20 gto 600 g AS/ha. For application of tubers or seed grain, dosages of 10mg to 1 g active substance per kg of seed grain or tubers may be used.

The contents of all references, patent applications and patents, citedthroughout this application are hereby expressly incorporated byreference. Each reference disclosed herein is incorporated by referenceherein in its entirety. Any patent application to which this applicationclaims priority is also incorporated by reference herein in itsentirety.

EXEMPLIFICATION OF THE INVENTION Example 1 Identification of Modulatorsof RNA Binding Proteins MEX-5, POS-1 and MEX-3

Expression and Purification of Recombinant MEX-5, POS-1, and MEX-3

MEX-5: The expression and purification of recombinant MEX-5 protein haspreviously been described in Pagano, J. M., Farley, B. M., McCoig, L. M.and Ryder, S. P. (2007) “Molecular basis of RNA recognition by theembryonic polarity determinant MEX-5;” J Biol Chem; 282, 8883-8894. Thefragment of mex-5 containing the TZF domain (amino acids 236-350) wasamplified from a commercially available ORFeome clone (Open Biosystems)and sub-cloned into the vector pMal-c (NEB), which encodes maltosebinding protein as an N-terminal fusion. This construct is termedpMal-MEX-5 (236-350).

MEX-5 is expressed in Escherchia. coli JM109 liquid cultures grown at37° C. Cultures were induced during mid log phase by the addition of 0.1mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) and allowed to grow forthree hours. An amount of 100 μM zinc acetate (final concentration) wasadded at the time of induction. The pelleted cells were resuspended inlysis buffer (50 mM Tris pH 8.8, 200 mM NaCl, 2 mM DTT, EDTA freeprotease inhibitor tablet (Roche), 100 μM Zn(OAc)₂) and lysed bysonication. The soluble protein was purified in three steps usingstandard chromatography methods: (1) amylose affinity (NEB), (2)Hi-TRAP-Q (GE Healthcare), and (3) Hi-TRAP-S (GE Healthcare). The Qbuffer was composed of 50 mM Tris, pH 8.8, 20 mM-1500 mM NaCl, 2 mM DTT,and 100 μM Zn(OAc)₂. The S buffer was composed of 50 mM MOPS, pH 6.0, 20mM-1500 mM NaCl, 2 mM DTT, 100 μM Zn(OAc)₂. The pure fractions,determined by coomassie-stained SDS-PAGE, were dialyzed into storagebuffer (20 mM Tris, pH 8.0, 25 mM NaCl, 100 μM Zn(OAc)₂, 2 mM DTT). Theprotein concentration was determined by measuring absorbance at 280 nmand the calculated extinction coefficient. The protein was stored at 50μM concentration at 4° C.

POS-1: The expression and purification of recombinant POS-1 protein hasbeen previously described in Farley, B. M., Pagano, J. M. and Ryder, S.P. (2008) “RNA target specificity of the embryonic cell fate determinantPOS-1.” RNA, 14, 2685-2697. The sequence encoding amino acids 80-180 ofPOS-1 was amplified from the corresponding ORFeome (Open Biosystems)clone via PCR and cloned in frame into the multiple cloning site ofpHMTc, a derivative of pMal-c2x (NEB) that includes an N-terminal 6-histag and a TEV protease site after MBP.

The protein was expressed from this construct in E. coli strain BL21(DE3) Gold (Stratagene). The protein expression was induced by additionof 1 mM IPTG and 100 μM Zn(OAc)₂ at mid-log phase. Cells were inducedfor three hours prior to harvesting and the cells were lysed andpurified using an amylose column (NEB). The eluate was passed through aHiTrap SP column, and the collected flow through was purified over aHiTrap Q (GE Healthcare) column. The following buffers were used: Lysisbuffer (50 mM Tris, pH 8.0, 200 mM NaCl, 2 mM DTT, 100 μM Zn(OAc)₂), Sbuffer (50 mM MOPS pH 6.0, 20 mM NaCl, 2 mM DTT, 100 μM Zn(OAc)₂), and Qbuffer (50 mM Tris, pH 8.8, 20 mM-750 mM NaCl, 2 mM DTT, 100 μMZn(OAc)₂). The purified POS-1 was dialyzed into storage buffer (25 mMTris-Cl pH 8.0, 25 mM NaCl, 100 μM Zn(OAc)₂, 2 mM DTT), concentrated toapproximately 100 μM using a 30,000 MWCO spin concentrator (vivascienceGroup), and stored at 4° C. for up to two months.

MEX-3: The sequence encoding amino acids 45-205 of MEX-3 was amplifiedfrom the ORFeome clone (Open Biosystems) and subcloned into pMal-c(NEB). MBP-MEX-3(45-205) was expressed and purified from BL21 (DE3) Gold(Stratagene) E. coli. A liquid culture grown at 37° C. was induced atmid-log phase with 1 mM isopropyl 1-thio-β-D-galactopyranoside and grownfor 3 hours before harvesting cells. The cells were lysed in lysisbuffer and purified using amylose affinity resin (NEB), followed by aHiTrap Q and Source 15Q (GE Healthcare) columns at 4° C. using the lysisbuffer and Q-buffer outlined for MEX-5, above. The purified MEX-3 wasdialyzed into storage buffer (25 mM Tris, pH 8.0, 25 mM NaCl, 2 mM DTT)and stored at 4° C. at a concentration between 30-50 μM.Fluorescein-Labeled RNA Oligonucleotides:

All RNA oligonucleotides were chemically synthesized by Dharmacon orIntegrated DNA Technologies. Upon arrival, each RNA was deprotected andstored according to the manufacturer's instructions. Prior to use, theRNA was resuspended in 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, and theconcentration determined by UV spectrophotometry using the calculatedextinction coefficient at 260 nm based on the specific sequence. Thesequence of the MEX-5 binding RNA (TCR2) isUUUCUUUAUAACUUGUUACAAUUUUUGAAA (SEQ ID NO:1). The POS-1 binding RNA(BMF018) is AACUAUUAUUAUUUGUUAUUCAUAUUUU (SEQ ID NO:2). The MEX-3binding RNA (SEQ14) is CGAGCAGGAAGUGUGCAGAGUUUAGGACGU (SEQ ID NO:3).

The RNA used for the small molecule screen was labeled at the 3′-endwith fluorescein by Dharmacon during synthesis. In a few cases, followup experiments were performed with post-synthetically labeled RNA. Inthis case, fluorescein 5-thiosemicarbazide (FTS C, Invitrogen) was usedto 3′-end label each RNA after treatment with sodium periodate (seeReines, S. A. and Cantor, C. R. (1974) “New fluorescent hydrazidereagents for the oxidized 3′-terminus of RNA” Nucleic Acids Res. 1,767-786). A representative 50 μl reaction consisted of 0.5 nanomolesRNA, 100 mM NaOAc, pH 5.1, and 5 nanomoles NaIO₄. After the reaction wascomplete (90 minutes at room temperature), the sample was ethanolprecipitated by adding 1 μl RNase free glycogen (Invitrogen 20 μg/μl),1/20^(th) volume of 5 M NaCl, and 2 volumes of 100% ice-cold ethanol.The resulting pellet was resuspended in 50 μl of 100 mM NaOAc, pH 5.1containing 1 mM FTSC and was allowed to react overnight at 4° C. in thedark. The unreacted label was removed by ethanol precipitation withresuspension of the pellet in a small volume of TE, and purificationover a Roche G-25 size exclusion spin column. The labeling efficiency isdetermined by calculating the ratio of fluorescein absorbance at 490 nmto RNA-fluorescein absorbance at 260 nm. Typical efficiencies are60-80%.

Screening Protocol:

MEX-5: The ability of MEX-5 to associate with RNA was monitored byfluorescence polarization using a victor V3 or victor V2V plate reader(Perkin Elmer) in 384-well FluoTrak microplates (Grenier). Each platecontained 320 wells with 32 no protein control wells and 32 no compoundcontrol wells. The concentration of MEX-5 and fluorescein-labeled TCR2RNA was chosen to maximize signal to noise while maintainingsub-saturation binding. The final concentration of reagents in eachexperimental well was 120 nM MBP-MEX-5, 2 nM TCR2 RNA, 192 μM testcompound, 50 mM Tris-HCl, pH 8.0, 100 mM NaCl 0.01% IGEPAL CA-360, 0.01mg/mL tRNA from S. cerevisiae (Sigma, 2000 Units) and 100 μM Zn(OAc)₂.The Z-value calculated for each plate from the no protein and nocompound controls ranged between 0.8 and 0.9 (see Zhang, J. H., Chung,T. D. and Oldenburg, K. R. (1999) “A Simple Statistical Parameter forUse in Evaluation and Validation of High Throughput Screening Assays” J.Biomol. Screen 4, 67-73).

Each day, two stock solutions were prepared, a protein mix and an RNAmix. The protein mix contained 200 nM MBP-MEX-5 dissolved in 100 μMZn(OAc)₂. The RNA mix contained 5 nM labeled TCR2 RNA, 125 mM Tris-HCl,pH 8.0, 250 mM NaCl, 0.025% IGEPAL CA-360 (Sigma), 0.025 mg/mL tRNA, and100 μM Zn(OAc)₂. A 96-pin liquid handling robot was used to transfer 2μL of 2 mM stock of test compound dissolved in DMSO into 320 wells of amicroplate. The remaining 84 wells were filled with 2 μL of DMSO (noprotein and no compound controls). Subsequently, a μFill plate dispenserwas used to transfer 30 μL of protein stock into the plate 352 wells ofthe plate. The remaining 32 wells received 30 μL of 100 μM Zn(OAc)₂ (noprotein controls). Finally, The μFill plate dispenser was used totransfer 20 μL of RNA stock into all 384 wells of the plate.

The apparent polarization (mP) of each well of the plate was determinedusing victor V3 or victor V2V plate reader. Each plate was measured intriplicate with a measurement time of 0.4 seconds per well, anexcitation band pass filter of 480±31 nm, and an emission band passfilter of 535±40 nm. The data was processed with Microsoft Excel andWavemetrics Igor Pro. The well score was defined by the followingequation:SCORE=(mP−mP_(average) _(—) _(no) _(—) _(protein) _(—)_(controls))/(mP_(average) _(—) _(no) _(—) _(drug) _(—)_(controls)−mP_(average) _(—) _(no) _(—) _(protein) _(—) _(controls))Compounds with a score of less than or equal to 0.25 (75% inhibition)where the fluorescence intensity (S) is within 2-fold of the controlwells were identified as compounds capable of modulating, e.g.,inhibiting, the RNA binding activity of MEX-5.POS-1: POS-1 inhibitor screening proceeded using the protocol describedabove, except MBP-POS-1 and BMF018 RNA were used in place of MEX-5 andTCR2 RNA.MEX-3: MEX-3 screening proceeded as above with the followingmodifications. MBP-MEX-3 protein and SEQ14 RNA were used in place ofMEX-5 and TCR2 RNA. The final concentration of MBP-MEX-3 was reduced to80 nM in each well, thus the concentration in the protein stock wasreduced to 133.3 nM. Zinc acetate was not used in the either the RNAstock or the Protein stock.

Results of the screening assays for each of MEX-5, POS-1 and MEX-3 areshown below in Table 2. Table 2 provides those test compounds which wereidentified as modulators, e.g., inhibitors, of the RNA binding activityof MEX-5, POS-1 and/or MEX-3, and includes the corresponding scores(where the lowest score correlates to the greatest inhibition) for eachcompound that were obtained in the assays for each of MEX-5, POS-1 andMEX-3.

TABLE 2 Compound Code Structure MEX-5 POS-1 MEX-3 A

−0.103068   0.0750083 — B

−0.0369119   0.0551393 — C

−0.0208829 −0.0247867 — D

−0.0158025 −0.0379222 — E

−0.0106525 −0.0161771 — F

−0.007108 −0.0278922 — G

−0.00640326 0.241935 — H

−0.00335626   0.0195217 — I

−0.00142058   0.0054316 — J

−0.000941405   0.085093 — K

  1.38E-05   0.0581118 — L

  0.0121396 −0.000863837 — M

  0.0162117   0.116787 — N

  0.0208867 — — O

  0.0229711   0.0476617 — P

  0.0341767   0.0430453 — Q

  0.0351486 — — R

  0.0351786 — — S

  0.050021   0.105408 — T

  0.0550846   0.0898578 — U

  0.0621123 — — V

  0.0626293 — — W

  0.064008   0.0539474 — X

  0.0668833 — — Y

  0.067749   0.0604342 — Z

  0.0687865   0.126705 — AA

  0.0713937   0.00702445 — AB

  0.0730459   0.108744 — AC

  0.0758265 — — AD

  0.0803921   0.0826922 — AE

  0.0810947   0.0360551 0.162354 AF

  0.081336   0.226509 — AG

  0.0861502 — — AH

  0.0875937   0.145547 — AI

  0.0912883   0.0249758 — AJ

  0.0923393 — — AK

  0.101513 — — AL

  0.103973   0.123145 — AM

  0.104822 — — AN

  0.105086 — — AO

  0.105831   0.0681423 — AP

  0.122397 — — AQ

  0.123778   0.115705 — AR

  0.129328 — — AS

  0.131352 — — AT

  0.132089 — — AU

  0.134432   0.247521 — AV

  0.140139   0.109312 — AW

  0.140504 — — AX

  0.141245 — — AY

  0.141274   0.180409 — AZ

  0.143526   0.253527 — BA

  0.151983   0.221567 — BB

  0.153606 — — BC

  0.154715 — — BD

  0.154759 — — BE

  0.160602 — — BF

  0.166567   0.0904628 — BG

  0.171728 — — BH

  0.176422 — — BI

  0.178688   0.106184 — BJ

  0.181913   0.11881 — BK

  0.183519 — — BL

  0.188022   0.275526 — BM

  0.188023 — — BN

  0.188711 — — BO

  0.190286 — — BP

  0.19239   0.0454242 — BQ

  0.195494 — — BR

  0.19904 — — BS

  0.200487   0.24294 — BT

  0.201886 — — BU

  0.208329   0.181499 — BV

  0.211271   0.122405 — BW

  0.211513 — — BX

  0.211718 — — BY

  0.213189   0.143999 — BZ

  0.214246 — — CA

  0.216367   0.230119 — CB

  0.21706   0.23927 — CC

  0.224919 — — CD

  0.226379   0.123733 — CE

  0.227469 — — CF

  0.229065 — — CG

  0.230436 — — CH

  0.230856 — — CI

  0.232018 — — CJ

  0.233565 — — CK

  0.233621 — — CL

  0.235106 — — CM

  0.236312 — — CN

  0.239373 — — CO

  0.240608   0.160345 — CP

  0.241146 — — CQ

  0.245197 — — CR

  0.24654 — — CS

  0.251831 — — CT

  0.252156 — — CU

  0.253312 — — CV

  0.255468 — — CW

—   0.0398467 — CX

—   0.0430453 — CY

—   0.0434891 — CZ

—   0.0488653 — DA

—   0.0651114 — DB

—   0.0689837 — DC

—   0.0749757 — DD

—   0.0750083 — DE

—   0.0886382 — DF

—   0.10079 — DG

—   0.108633 — DH

—   0.118649 — DI

—   0.119337 — DJ

—   0.1383 — DK

—   0.142516 — DL

—   0.1431 — DM

—   0.143623 — DN

—   0.166782 — DO

—   0.166873 — DP

—   0.167907 — DQ

—   0.171713 — DR

—   0.171815 — DS

—   0.176583 — DT

—   0.177235 — DU

—   0.180302 — DV

—   0.184556 — DW

—   0.189883 — DX

—   0.196968 — DY

—   0.200285 — DZ

—   0.20137 — EA

—   0.203009 — EB

—   0.209605 — EC

—   0.217393 — ED

—   0.220348 — EE

—   0.225108 — EF

—   0.228172 — EG

—   0.229412 — EH

—   0.230119 — EI

—   0.230383 — EJ

—   0.233942 — EK

—   0.236507 — EL

—   0.237867 — EM

—   0.241791 — EN

—   0.247036 — EO

—   0.253668 — EP

—   0.259513 — EQ

—   0.26035 — ER

—   0.261571 — ES

—   0.26453 — ET

—   0.265118 — EU

—   0.26601 — EV

—   0.274806 — EW

—   0.275235 — EX

— — 0.0673989 EY

— — 0.0896497 EZ

— — 0.123959 FA

— — 0.209341 FB

— — 0.258457

Further screenings were performed on select compounds of the invention,as described above, and the results are shown in Table 3.

TABLE 3 Dose Response MEX-5 Dose Response POS-1 Compound (μM) (μM) E 1.50.32 BR 39 820 BK 540 ND DG 160 ND DD ND 300 DJ 3.5 ND C 33 7 BO 75 16

Example 2 Hermaphrodite Worm Reproduction Assay

In order to further evaluate the ability of a particular RNA bindingmodulatory compound identified herein to inhibit embryogenesis, e.g., ina nematode, the compounds identified herein are tested in a standardhermaphrodite worm reproduction assay s. Larval worms are hatchedovernight into medium containing the compound in an agar plate. Then,feeder bacteria is added to each plate and the worms cultured until theybegin to produce eggs. Adult worms are removed or killed. Finally, theratio of dead eggs to hatchlings is determined by inspection with astereomicroscope.

Example 4 Dose Response Gel Shift Assays

A further dose response assay was performed on select compounds of theinvention as described below. A sub-saturating concentration of MEX-5 orPOS-1 (120 nM) was equilibrated with limiting fluorescein labeled RNA(2-3 nM, TCR2 RNA or MEX-3 UTR fragment RNA respectively) in thepresence of varying concentrations of compound. Following equilibration,the reactions were loaded onto a 5% slab polyacrylamide gel andsubjected to electrophoresis for about 1 hour to separate protein-RNAcomplex from free RNA. The gel was scanned on a FUJI FLA-5000 imager,and the fraction of bound RNA determined by dividing the intensity ofthe bound RNA by the intensity of the bound RNA plus the free RNA. Thefraction of the compound bound to the protein was plotted as a functionof compound concentration and fit to a sigmoidal dose response functionin order to determine the IC₅₀, the concentration that gives halfmaximal inhibition. The results are seen in Table 4.

TABLE 4 Gel Shift MEX-5 Gel Shift POS-1 Compound (μM) (μM) E 2.0 0.17 BR78 150 BK 110 ND DG 170 ND DD ND 260 DJ 3.8 ND C 9.5 5.1 BO 120 33

Example 5 C. elegans Viability/Sterility Assay

In order to determine whether select compounds of the inventioninhibited viability of embryos, Caenorhabditis elegans N2 strain wormswere cultured on NGM agar plates using OP50 strain Escherchia coli asfood. Young adult worms with a single row of embryos were selected formicroinjection. Each worm was mounted on a dried agarose paddedcoverslip in halocarbon oil. Varying concentrations of compound, or aDMSO control, were microinjected into each gonad arm of the worm usingan inverted microscope with DIC optics. Injected worms were then allowedto recover for 2-3 hours at 15° C. Approximately 10-40 worms wereinjected per compound tested. Individual worms that survivedmicroinjection were singled out onto new NGM agar plates and allowed torecover overnight at 15° C. The plates were moved to room temperaturethe next morning. At the end of the day, the adult worm was transferredto a new plate. The embryos laid on each plate were monitored forhatching and inspected for unusual phenotypes. Nematode fertility wasestimated by counting the total number of progeny on both plates.Viability was estimated by monitoring the extent of hatching. Theresults of this assay using compound E are shown below in Table 5.

TABLE 5 Day 1 Day 2 Worm No. Embryos Hatchlings Embryos Hatchlings 1 034 0 0 2 0 2 0 0 3 2 6 0 0 4 0 4 0 0 5 0 67 0 0 6 1 11 0 0

The results of this assay indicate that compound E may sterilize wormsat a stage prior to fertilization. The worms seemed to lose fertilityover the course of two days. In contrast, 27 worms were injected withDMSO controls, 10 survived injection, and they continued to produce eggsafterwards.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the following claims.

1. A method for treating or preventing a parasitic associated state in asubject comprising administering to said subject an effective amount ofcompound E:

or a pharmaceutically acceptable salt thereof, such that said parasiticassociated state is treated or prevented, wherein compound E modulatesthe RNA binding activity of an RNA binding protein selected from a groupconsisting of POS-1 and MEX-5.
 2. The method of claim 1, wherein saidsubject is a plant, an animal or a human.
 3. The method of claim 1,wherein said parasitic associated state is a parasitic infestation orparasitic re-infestation or a disease caused by a parasitic infestation.4. The method of claim 1, wherein said method includes protecting plantsfrom a parasitic infestation, inhibiting embryogenesis in a parasite orin a subject suffering from a parasitic infestation, or reducingparasitic burden in soil, in plants or in an animal suffering from aparasitic infection.
 5. The method of any one of claims 1, 2, 3 and 4,wherein said parasite is a helminth.
 6. The method of claim 5, whereinsaid helminth is selected from the group consisting of a cestode, atrematode and a nematode.
 7. A method for inhibiting embryogenesis in aparasite, comprising contacting said parasite with an effective amountof compound E:

or a pharmaceutically acceptable salt thereof, such that saidembryogenesis is inhibited, wherein compound E modulates the RNA bindingactivity of an RNA binding protein selected from a group consisting ofPOS-1 and MEX-5.
 8. The method of claim 7, wherein the parasite ispresent in a subject.
 9. The method of claim 7 or 8, wherein saidparasite is a helminth.
 10. The method of claim 9, wherein said helminthis selected from the group consisting of a cestode, a trematode and anematode.
 11. A method for treating or preventing an inflammatorydisorder caused by a parasitic associated state in a subject comprisingadministering to said subject a therapeutically effective amount ofcompound E:

or a pharmaceutically acceptable salt thereof, such that saidinflammatory disorder is treated or prevented, wherein compound Emodulates the RNA binding activity of an RNA binding protein selectedfrom a group consisting of POS-1 and MEX-5.
 12. The method of claims 1,7 and 11, wherein the RNA binding protein is required for embryogenesis.13. A pharmaceutical composition comprising a therapeutically effectiveamount of compound E:

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, wherein compound E modulates the RNA bindingactivity of an RNA binding protein selected from a group consisting ofPOS-1 and MEX-5.
 14. A composition comprising a pesticidally effectiveamount of compound E:

and an agronomically acceptable carrier, wherein compound E modulatesthe RNA binding activity of an RNA binding protein selected from a groupconsisting of POS-1 and MEX-5.
 15. A method for treating or preventing aparasitic associated state in a subject comprising administering to saidsubject an effective amount of compound DI:

or a pharmaceutically acceptable salt thereof, such that said parasiticassociate state is treated or prevented, wherein compound DI modulatesthe RNA binding activity of an RNA binding protein POS-1.
 16. The methodof claim 15, wherein said subject is a plant, an animal or a human. 17.The method of claim 15, wherein said parasitic associated state is aparasitic infestation or parasitic re-infestation or a disease cause bya parasitic infestation.
 18. The method of claim 15, wherein said methodincludes protecting plants from a parasitic infestation, inhibitingembryogenesis in a parasite or in a subject suffering from a parasiticinfestation, or reducing parasitic burden in soil, in plants or in ananimal suffering from parasitic infection.
 19. The method of any one ofclaims 15-18, wherein said parasite is a helminth.
 20. The method ofclaim 19, wherein said helminth is selected from the group consisting ofa cestode, a trematode and a nematode.
 21. A method for inhibitingembryogenesis in a parasite, comprising contacting said parasite with aneffective amount of compound DI:

or a pharmaceutically acceptable salt thereof, such that saidembryogenesis is inhibited, wherein compound DI modulates bindingactivity of an RNA binding protein POS-1.
 22. The method of claim 21,wherein the parasite is present in a subject.
 23. The method of claim 21or 22, wherein said parasite is a helminth.
 24. The method of claim 23,wherein said helminth is selected from the group consisting of acestode, a trematode and a nematode.
 25. A method for treating orpreventing an inflammatory disorder caused by a parasitic associatedstate in a subject comprising administering to said subject atherapeutically effective amount of compound DI:

or pharmaceutically acceptable salt thereof, such that said inflammatorydisorder is treated or prevented, wherein compound DI modulates the RNAbinding activity of an RNA binding protein POS-1.
 26. The method ofclaims 15, 21 and 25, wherein the RNA binding protein is required forembryogenesis.
 27. A pharmaceutical composition comprising atherapeutically effective amount of compound DI:

or pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, wherein compound DI modulates the RNA bindingactivity of an RNA binding protein POS-1.
 28. A composition comprising apesticidally effective amount of compound DI:

and an agronomically acceptable carrier, wherein compound DI modulatesthe RNA binding activity of an RNA binding protein POS-1.